Food Byproducts as a New and Cheap Source of Bioactive

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Journal of Agricultural and Food Chemistry

Food By-products as a New and Cheap Source of Bioactive Compounds: Lignans with Antioxidant and Anti-inflammatory Properties from Crataegus pinnatifida Seeds Xiao-Xiao Huang†,‡, Ming Bai†,‡, Le Zhou†,‡, Li-Li Lou†,‡, Qing-Bo Liu†,‡, Yan Zhang§, Ling-Zhi Li†,‡, Shao-Jiang Song*,†,‡

†

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,

Shenyang 110016, PR China; ‡

Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of

Education, Shenyang Pharmaceutical University; §

School of Pharmaceutical engineering, Shenyang Pharmaceutical University.

*Correspondence author Prof. Dr. Shao-Jiang Song School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang Liaoning 110016, PR China Phone: +86-24-23986088 Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT:

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During the process of manufacturing hawthorn juice and jam, a significant

3

quantity of by-products (leaves, seeds) are generated. The antioxidant and

4

anti-inflammatory bioassay-guided fractionation of the extract of Crataegus

5

pinnatifida (hawthorn) seeds has led to the isolation of eight new lignans,

6

hawthornnin A−H (1−8) and seven known analogs (9−15). Their structures were

7

elucidated by spectroscopic techniques, including 1D and 2D NMR and CD spectra.

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The radical scavenging effects of all isolated compounds were investigated. 1−6 and 8

9

showed moderate activity against 2,2-diphenyl-1-pikrylhydrazyl (DPPH) while 1−6

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and 14 displayed good 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)

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(ABTS) free radical scavenging activities that was even more potent than trolox. In

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addition, all isolates were evaluated for their anti-inflammatory activities by detecting

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the nitric oxide (NO) and tumor necrosis factor α (TNF-α) production by the

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LPS-induced murine macrophage cell line RAW264.7, compounds 1−7, 13 and 14

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exhibited potent inhibition of NO and TNF-α production. The structure-activity

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relationships of isolated lignans were also examined and the results obtained show

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that C. pinnatifida seeds can be regarded as a potential new and cheap source of

18

antioxidants and inflammation inhibitors.

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KEYWORDS: Crataegus pinnatifida, hawthorn, food by-product, seeds, lignans,

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antioxidant, anti-inflammatory

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INTRODUCTION

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Reactive oxygen species (ROS) play a key role in the pathological processes

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associated with a variety of various serious diseases, such as atherogenesis,

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neurodegeneration, cancer and chronic inflammation.1,2 Inflammation is recognized as

27

a biological response to tissue injury and severe inflammation contributes to many

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inflammatory disorders.3 Recent studies have produced significant evidence that ROS

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are closely involved in the pathogenesis of inflammatory processes.4 There is a link

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between radical scavenging activities and anti-inflammatory effects and, therefore,

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antioxidants play a key role in the treatment of inflammatory diseases.5,6

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In recent years, there has been increasing interest in identifying new possible

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sources of natural antioxidants and other health promoting compounds.7 Many natural

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bioactive constituents, including phenols from vegetables, fruits or grains, have been

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used experimentally as effective protection against ROS or inflammation-related

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tissue damages.8 Phenols are non-nutritive constituents produced by secondary

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metabolism in plants, which including several classes of phenolic acids, flavonoids,

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lignans, condensed tannins, and stilbenoids.9 Many by-products created during food

39

production, such as skins and seeds, contain phenols with potential applications as

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natural antioxidants to prevent diseases.10,11

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Crataegus pinnatifida (family Rosaceae), also referred to as “Hawthorn”, is

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associated with more than 280 species. Hawthorn is commonly distributed in China,

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Europe, and North America.12-14 In China, hawthorn fruit has been used as a raw 3



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material for functional foods, and has also been included in the Chinese Pharmacopeia

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as a traditional Chinese medicine. 15-16 Some studies have reported that hawthorn has

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the capacity to scavenge free radicals and inhibit the oxidation of lowdensity

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lipoprotein (LDL), as well as having anti-inflammatory activities.17-19

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Hawthorn is mainly for consumed as fresh fruit or processed juice and jam and

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the manufacturing of hawthorn juice and jam results in a significant quantity of

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by-products, such as leaves and seeds. Most of them are currently treated as industrial

51

waste or used as animal feed or fertilizer. Thus, their potential use has attracted our

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attention and the antioxidant and anti-inflammatory bioassay-guided fractionation of

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the extract of C. pinnatifida (hawthorn) seeds has led to the isolation of eight new

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lignans (1-8) and seven known lignans (9-15). In the present study, we describe the

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isolation and structural investigation of these newly isolated compounds, as well as

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the evaluation of their antioxidant capacity and inhibitory effects on LPS-induced NO

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and TNF-α production in mouse macrophage RAW264.7 cells. This study will help us

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to identify newer anti-inflammatory and anti-oxidant constituents of C. pinnatifida

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seeds and help in the use of this by-product generated in food processing to improve

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human health.

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MATERIALS AND METHODS

62 63

Chemicals

and

Reagents.

Minocycline,

silybin,

6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic 4


acid

DPPH,

ABTS, (trolox),

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lipopolysaccharide (LPS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) were purchased from Sigma-Aldrich. Cell culture medium

66

[Dulbecco’s modified Eagle’s medium (DMEM)], fetal bovine serum (FBS),

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penicillin, streptomycin, and all other materials required for culturing cells were

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purchased from Gibco BRL, Life Technologies.

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Apparatus. Optical rotations were determined using a JASCO P-1020 polarimeter.

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The UV spectra were recorded on a Shimadzu UV-1700 spectrometer and the CD

71

spectra were obtained using a MOS 450 detector from BioLogic. The FT-IR spectra

72

were measured using a Bruker IFS-55 spectrometer. NMR spectra were performed on

73

a Bruker AV-600 spectrometer. HRESIMS experiments were measured using an

74

Agilent G6520 Q-TOF spectrometer. Preparative and semipreparative RP-HPLC

75

isolation was achieved with an Agilent 1100 and 1200 instrument using an Acchrom

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XAqua C18 column (5 µm, 250 mm × 20 mm) and a YMC C18 column (5 µm, 250

77

mm × 10 mm). Peaks were identified using a refractive index detector (RID). The

78

absorbances in bioassays were measured using a varioskan flash multimode reader

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(Thermo scientific). Column chromatography (CC) was performed using silica gel

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(200-300 mesh; Qingdao Marine Chemical Inc.), macroporous adsorption resin D101

81

(Cangzhou Bon Adsorber Technology Co., Ltd) and ODS (50 µm, YMC Co. Ltd).

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TLC was conducted on precoated glass plates (SiO2 GF254; Qingdao Marine

83

Chemical Inc.).

84

Plant material. The seeds of C. pinnatifida were collected in Hebei province, P. R. 5



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China, in June 2011, and were identified by Professor Jin-Cai Lu (School of

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Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A

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voucher specimen (No. 20110701) has been deposited in the Herbarium of Shenyang

88

Pharmaceutical University, Liaoning, P. R. China.

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Extraction and isolation. The air-dried seeds of C. pinnatifida (30 kg) were

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crushed and refluxed with 70% EtOH (3 × 30 L) for 4 h. The solvent was filtered,

91

concentrated under vacuum and, then, the extract (1500 g) was suspended in H2O and

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partitioned sequentially with ethyl acetate and n-BuOH. The ethyl acetate and n-butyl

93

alcohol fractions were tested in antioxidant (scavenging activities for DPPH and

94

ABTS•+) and anti-inflammatory assays, and the ethyl acetate extract was found to

95

exhibit stronger activities. The ethyl acetate extract (420 g) was suspended in H2O and

96

then chromatographed on a D101 macroporous resin column using H2O−EtOH (from

97

100:0 to 5:95) as eluents, yielding four fractions (Fractions A−D). The four fractions

98

were also tested in antioxidant and anti-inflammatory assays, and the fraction B

99

(H2O:EtOH = 70:30; 128.0 g) was found to exhibit more potent activities.

100

The fraction B was subjected to silica gel CC and eluted with CH2Cl2−CH3OH to

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yield eight fractions (B1−B8). Among of them, B3 (20.2 g) was further purified by

102

ODS CC using H2O-MeOH as a mobile phase gradient (from 95:5 to 50:50) to afford

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five fractions (B3-1−B3-5) based on HPLC analysis. B3-3 (3.4 g) was subjected to

104

further silica gel column chromatography and eluted with CH2Cl2:CH3OH (from 95:5

105

to 80:20) to yield 8 fractions (B3-3-1−B3-3-8) based on silica gel TLC analysis. B3-3-5 6


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was subjected to preparative HPLC and eluted with CH3OH-H2O (40:60) to yield two

107

fractions (B3-3-5-1 and B3-3-5-2). B3-3-5-1 was subjected to semipreparative HPLC and

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eluted with CH3CN-H2O (23:77) to yield 1 (18 mg, tR 31 min, 4 mL/min) and 3 (8 mg,

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tR 36 min, 4 mL/min). B3-3-5-2 was subjected to semipreparative HPLC and with

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CH3CN-H2O (23:77) as eluents to afford 2 (8 mg, tR 29 min, 4 mL/min) and 4 (6 mg,

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tR 39 min, 4 mL/min). B3-4 was also subjected to silica gel column chromatography

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and eluted with CH2Cl2:CH3OH (from 95:5 to 80:20) to afford 9 fractions

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(B3-4-1−B3-4-9) based on silica gel TLC analysis. B3-4-4 was subjected to semipreparative

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HPLC and eluted with CH3CN-H2O (30:70) to yield 5 (4 mg, tR 27 min, 4 mL/min)

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and 6 (15 mg, tR 28 min, 4 mL/min). B3-2 was separated by silica gel column and

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eluted with CH2Cl2:CH3OH to afford 7 fractions (B3-2-1−B3-2-7). B3-2-3 was subjected to

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semipreparative HPLC and with CH3CN−H2O (22:78) as eluents to give 13 (16 mg, tR

118

40 min, 4 mL/min), 14 (20 mg, tR 29 min, 4 mL/min), 15 (11 mg, tR 21 min, 4

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mL/min) and 7 (8 mg, tR 33 min, 4 mL/min). B3-2-5 was subjected to semipreparative

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HPLC with CH3OH−H2O (19:81) as eluents to yield 8 (4 mg, tR 52 min, 4 mL/min).

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B3-2-5 was subjected to semipreparative HPLC and eluted with CH3CN−H2O (9:91) at

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to give 9 (42 mg, tR 18 min, 4 mL/min), 10 (40 mg, tR 19 min, 4 mL/min), 11 (66 mg,

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tR 23 min, 4 mL/min) and 12 (68 mg, tR 24 min, 4 mL/min).

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Hawthornnin A (1): yellow oil; [α]20 D -7.0 (c 0.10, MeOH); UV (MeOH) λmax

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(logε): 233 (0.63), 283 (0.31); CD [c 0.20 mg/mL, MeOH, nm (ε)]: 234 nm (9.02),

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256 nm (-7.72), 283 nm (-9.97); IR (KBr) νmax: 3384, 2938, 1605, 1516, 1463, 1141, 7



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1032, 822 and 760 cm-1; HRESIMS: m/z 535.1902 [M + Na]+ (calcd for C28H32O9Na,

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535.1939); 1H and 13C NMR: see Tables 1 and 2.

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Hawthornnin B (2): yellow oil; [α]20D +3.5 (c 0.13, MeOH); UV (MeOH) λmax

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233 nm (0.86), 284 nm (1.52); IR (KBr) νmax: 3407, 2928, 1610, 1510, 1453, 1243,

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133


134

Hawthornnin C (3): yellow oil; [α]20 D -4.5 (c 0.12, MeOH); UV (MeOH) λmax

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(logε): 227 (0.73), 281 (0.33); CD [c 0.15 mg/mL, MeOH, nm (∆ε)]: 237 nm (3.45),

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267 nm (-10.26), 279 nm (-11.02), 286 nm (-7.01); IR (KBr) νmax: 3357, 2938, 1604,

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1517, 1464, 1276, 1217, 822 and 765 cm-1; HRESIMS: m/z 535.1941 [M + Na]+

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(calcd for C28H32O9Na, 535.1939); 1H and 13C NMR: see Tables 1 and 2.

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Hawthornnin D (4): yellow oil; [α]20D +7.2 (c 0.10, MeOH); UV (MeOH) λmax

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141

245 nm (2.02), 278 nm (3.44); IR (KBr) νmax: 3406, 2936, 1605, 1498, 1452, 1274,

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143


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Hawthornnin E (5): yellow oil; [α]20 D +4.2 (c 0.16, MeOH); UV (MeOH) λmax

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242 nm (2.58), 281 nm (1.26); IR (KBr) νmax: 3382, 2940, 1603, 1518, 1432, 1274,

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1030, 819 and 769 cm-1; HRESIMS: m/z 549.2064 [M + Na]+ (calcd for C29H34O9Na, 8


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Hawthornnin F (6): yellow oil; [α]20D -8.7 (c 0.13, MeOH); UV (MeOH) λmax

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(logε): 226 (0.73), 281 (0.38); CD [c 0.15 mg/mL, MeOH, nm (ε)]: 234 nm (-7.52),

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283 nm (-4.87); IR (KBr) νmax: 3405, 2969, 1603, 1518, 1274, 1124, 1031, 819 and

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769 cm-1; HRESIMS: m/z 549.2057 [M + Na]+ (calcd for C29H34O9Na, 549.2095); 1H

153

and 13C NMR: see Tables 1 and 2.

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Hawthornnin G (7): yellow oil; [α]20 D -5.8 (c 0.11, MeOH); UV (MeOH) λmax

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261 nm (3.78), 282 nm (-0.91); IR (KBr) νmax: 3357, 2940, 1667, 1599, 1512, 1133,

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158


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Hawthornnin H (8): yellow oil; [α]20 D -6.5 (c 0.10, MeOH); UV (MeOH) λmax

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(logε): 230 (0.63), 280 (0.28); CD [c 0.10 mg/mL, MeOH, nm (ε)]: 236 nm (-5.72),

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281 nm (-0.84); IR (KBr) νmax: 3381, 2904, 1604, 1514, 1422, 1267, 1135, 1030, 817

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and 751 cm-1; HRESIMS: m/z 445.1458 [M + Na]+ (calcd for C21H26O9Na, 445.1469);

163

1

H and 13C NMR: see Tables 1 and 2.

164

Assay for DPPH radical scavenging activity. The DPPH scavenging activity

165

assay was adopted from a previous report.20 The 0.1 mM solution of DPPH in ethanol

166

was prepared firstly, and then 100 µL of this solution was mixed with 100 µL of

167

sample solution in a 96-well microplate. After incubating for 30 min in the dark at

168

37 °C, the absorbance was measured using a varioskan flash multimode reader at 517 9



169

nm. The percentage of scavenged DPPH was calculated using the following equation:

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DPPH scavenging activity (%) = [1 − (S − Sb) / (C − Cb)] × 100%. where S, Sb, C and

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Cb are the absorbances of the sample, blank sample, control and blank control,

172

respectively. All experiments were performed in triplicate and trolox was used as a

173

positive control.

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Assay for ABTS radical scavenging activity. The ABTS radical scavenging

175

activity of isolates was determined as in a previous report with some modifications.20

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ABTS radical cations (ABTS•+) were produced by reacting a 7 mM stock solution of

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ABTS with 2.45 mM potassium persulfate and allowing the mixture to stand in the

178

dark at room temperature for 12 h before use. The ABTS•+ solution was then diluted

179

with ethanol to the absorbance of 0.7 ± 0.05 at 734 nm. Different concentrations of

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test samples (100 µL) and ABTS•+ solution (150 µL) were added to each well of the

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96-well plates. After incubating at 37 °C for 30 min, the absorbance at 734 nm was

182

recorded and the percentage of ABTS free radical scavenging activity was calculated

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using the same formula as for the DPPH assay.

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Cell culture and cell viability assay. Mouse macrophage RAW264.7 cells were

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cultured in DMEM, 100 mg/mL streptomycin, and 100 U/mL penicillin. Cells were

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plated at a density of 5 × 105/mL and then cultured in a 96-well plate containing

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DMEM which supplemented with 10% FBS for 24 h until almost confluent. Cell

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viability was evaluated by MTT assay. Cells were treated with test samples (100 µM)

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for 1 h and stimulated with LPS (100 ng/mL) for 24 h, and then, the cells were 10


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incubated with MTT (0.25 mg/mL) followed by incubation for 4 h at 5% CO2, 37 °C.

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The formation of formazan was recorded at 540 nm using a varioskan flash

192

instrument.21,22

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NO assay in RAW264.7 cells. NO produced by the cells was determined by

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assaying the levels of NO2- using the Griess reagent. 22 RAW264.7 cells were plated at

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a density of 5 × 105/mL in a 96-well plate and pre-treated with test samples at

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concentration of 100, 50, 10 and 1 µM, respectively. After the cells were incubated

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with samples and stimulated with LPS (100 ng/mL) for 24 h, the cell culture media

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were collected to determinate NO. Briefly, a 100 µL aliquot of each sample was added

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to an equal volume of Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1%

200

N-naphtyl-ethylenediamine dihydrochloride) in a 96-well plate, and then incubated at

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37 °C for 10 min. After incubation, the absorbance was recorded at 540 nm on a

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varioskan flash instrument. The amount of NO in the sample was calculated using

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NaNO2 standard curve and minocycline was used as a positive control.

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TNF-α assay in RAW264.7 cells. The levels of TNF-α were determined using

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ELISA kit (R&D company) according to the manufacturer's instructions. TNF-α was

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determined from a standard curve and silybin was used as positive control. 23

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Statistical analysis. Results were expressed as mean ± standard error of means

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(S.E.M) of three determinations. The IC50 values were calculated using Microsoft

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Excel software. One-way analysis of variance (ANOVA) followed by Dunnett’s test

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was used for statistical analysis. 11



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RESULTS AND DISCUSSION

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The HRESIMS of 1 revealed the molecular formula of C28H32O9 from the

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pseudomolecular ion at m/z 535.1902 [M + Na]+. The UV spectrum showed the

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absorbances at λmax 233 and 283 nm and the IR spectral data indicated the presence of

215

OH (3384 cm-1) and an aromatic ring (1605, 1516 and 1463 cm-1). Eight aryl proton

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signals [δH 6.89 (d, J = 1.6 Hz, H-2), 6.75 (d, J = 8.1 Hz, H-5), 6.77 (dd, J = 8.1, 1.6

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Hz, H-6), 6.51 (br.s, H-2′), 6.54 (br.s, H-6′), 6.43 (d, J = 1.8 Hz, H-2′′), 6.61 (d, J =

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8.1 Hz, H-5′′), 6.50 (d, J = 8.1, 1.8 Hz, H-6′′)] found in the 1H NMR spectrum of 1

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(Table 1) were attributed to two 1,3,4-trisubstituted (ABX system) moieties, and one

220

1,3,4,5-tetrasubstituted (AX system) phenyl moiety. In addition, signals for four

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aliphatic methine protons [δH 5.49 (d, J = 5.5 Hz, H-7), 3.40 (m, H-8), 4.32 (d, J = 8.6

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Hz, H-7′) and 2.96 (m, H-8′)], four methoxy protons [δH 3.79 (3H, s, OCH3-3), 3.72

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(3H, s, OCH3-3′), 3.64 (3H, s, OCH3-3′′) and 3.24 (3H, s, OCH3-7′)], and two sets of

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oxygenated methylene protons [δH 3.67 (dd, J = 11.0, 5.1 Hz, H-9a), 3.48 (dd, J =

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11.0, 8.3 Hz, H-9b), 4.07 (dd, J = 10.9, 5.0 Hz, H-9′a) and 3.92 (dd, J = 10.9, 7.1 Hz,

226

H-9′b)] were observed. The

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characteristic of four methoxy carbons, 18 phenyl carbons and six aliphatic carbons.

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In addition to the above spectroscopic findings, the HMBC spectrum of 1 showed

229

cross-peaks between H-7/C-1, C-2, C-6, C-9, C-4′, C-5′, and H-7′/C-1′, C-2′, C-6′,

230

C-9′, C-1′′. Moreover, other long-range correlations, including H-8/C-4′, C-5′, and

231

H-8′/C-1′′, C-2′′, C-6′′, were also found (Fig. 2). Thus, the postulated structure of 1

13

C NMR spectrum (Table 2) showed 28 signals,

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contains two phenylpropanoid moieties and one benzene moiety. Subtracting nine

233

double bonds from the 13 degrees of unsaturation suggested that four rings remained

234

in 1. Together with the above findings, 1 was proposed to have a

235

dihydrobenzo[b]furan neolignan skeleton, which is similar to that of the

236

dehydrodiconiferyl alcohol. Furthermore, long-range correlations between H-7′/C-1′′,

237

H-9′/C-1′′, and H-8′/C-2′′, 6′′, as well as the other correlated cross-peaks found in the

238

HMBC spectrum, supported the presence of a C-C linkage between the

239

dehydrodiconiferyl moiety and a 3-methoxy-4-hydroxy-benzene moiety (Fig. 2).24 In

240

addition, the coupling constant between H-7 and H-8 was 5.5 Hz (in CD3OD) in the

241

1

242

in the NOESY spectrum in 1, suggested the relative conformation of H-7 and H-8 as

243

trans. The CD spectrum showed a negative Cotton effect (ε283 -9.97), so it was found

244

that 1 had the 7R,8S-configuration.25,26 The threo relative configuration at the

245

C-7′/C-8′ in 1 was suggested by comparing the 13C NMR spectra with those of threo

246

and erythro isomers.27,28 Thus, the structure of 1 was established and it was named

247

hawthornnin A.

248

H NMR spectrum, and cross-peaks of H-7/H-9, H-8/H-2 and H-8/H-6 were observed

Compound 2 was assigned as C28H32O9 from its HRESIMS data, and its UV, IR,

249

1

H NMR and 13C NMR spectroscopic data were identical with those of compound 1,

250

which suggested the planar structure of 2 was the same as 1. The trans relative

251

configuration at C-7 and C-8 was confirmed by the J7,8 value of 6.0 Hz in the 1H

252

NMR spectrum and the existence of cross-peaks (H-7/H-9, H-8/H-2 and H-8/H-6) in 13



253

the NOESY spectrum. Furthermore, the CD cotton curve (ε284 1.52) of 2 was opposite

254

to that of 1, indicating the configuration of the dihydrofuran ring was 7S,8R.25,26 In

255

addition, the chemical shifts of C-7′ and C-8′ indicated compound 2 also had a

256

7′,8′-threo-configuration.27,28 Thus, compound 2 was identified as an optical isomer of

257

1 and given the trivial name hawthornnin B.

258

Compound 3 was also identified as C28H32O9 by HRESIMS at m/z 535.1941 [M

259

+ Na]+. Its 13C NMR data were very similar to 1 except for a small difference in the

260

chemical shifts of C-7′ (∆δ 2.7), and a small coupling constant of H-7′ (J = 5.2 Hz)

261

was observed in the 1H NMR spectrum, which was different from that of 1, and

262

indicated that 3 had a 7′,8′-erythro-configuration.27,28 In addition, the 7,8-trans

263

configuration of 3 was obtained from the J7,8 value (5.8 Hz) and the NOESY spectrum.

264

The positive Cotton effect at 278 nm in the CD spectrum supported the

265

7S,8R-configuration.25,26 Therefore, the structure of 3 was identified as shown in Fig.

266

1 and had been accorded the trivial name hawthornnin C.

267

The UV, IR and NMR spectra of 4 were identical to those of 3, suggesting that

268

the planar structure of 4 was the same as 3. The chemical shifts of C-7′ and C-8′

269

suggested a 7′,8′-erythro relative configuration.27,28 In addition, the relative

270

configuration of C-7/C-8 was suggested to be relative-trans according to the J7,8 value

271

of 6.3 Hz. This was also verified by NOE correlations of H-7/H2-9 and H-8/H-6 and

272

H-8/H-2 in the NOESY spectrum. The positive CD effect at 278 nm of 4, supported

273

the 7R,8S-configuration25,26 and compound 4 was named hawthornnin D. 14


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Compound 5 was obtained as a yellow oil, with C29H34O9 as its molecular

275

formula, as indicated by HRESIMS at m/z 549.2064 [M + Na]+. The IR spectrum

276

displayed absorption bands for OH (3382 cm-1) and aromatic ring (1603 and 1518

277

cm-1) groups. The 1H and

278

except for the presence of the OCH3-7′ (δH 3.24; δC 57.0), which was replaced by an

279

ethoxy group (δH 3.30, 1.08; δC 65.2, 15.7) in compound 5. These minor structural

280

changes were further supported by HMQC and HMBC correlations (Fig. 2). The

281

erythro configuration between the two chiral centers at C-7′ and C-8′ was determined

282

by comparing the NMR spectra with those of the erythro and threo isomers.27,28 The

283

trans relationship between H-7 and H-8 was inferred from their coupling constant (J7,8

284

= 6.1 Hz), which was also further verified by the NOE correlations between H-7 and

285

H2-9 and between H-8 and H-2, 6. The positive CD effect at 281 nm of 5 supported

286

the 7S,8R-configuration 25,26 and it was named hawthornnin E.

13

C NMR spectra of 5 showed similarities to those of 1,

287

The molecular formula of 6 was determined as C29H34O9 by the [M + Na]+ ion

288

peak at m/z 549.2057 in the HRESIMS. Its IR and NMR spectroscopic data were in

289

good agreement with those of 5 (Tables 1 and 2), suggesting that the planar structure

290

and relative configuration of 6 was the same as that of compound 5. The CD spectrum

291

showed a negative Cotton effect (ε283 -4.87), which indicated that the absolute

292

configuration of the dihydrofuran ring was 7R,8S.25,26 Consequently, the structure of 6

293

was fully identified and it was named hawthornnin F.

294

Compound 7 gave a positive HRESIMS m/z 395.1469 [M + Na]+ (calcd. for 15



295

C21H24O6Na, 395.1465), consistent with the molecular formula C21H24O6. The IR

296

spectrum of 7 showed absorption for hydroxyl (3357 cm-1) and aromatic (1599 and

297

1512 cm-1) functions. The NMR data of 7 (Tables 1 and 2) were in good agreement

298

with those of 2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-5-(3-methoxyallyl)-

299

7-methoxycoumaran.29 The relative configuration of compound 7 was suggested to be

300

relative-trans according to the J7,8 value of 6.6 Hz. In addition, the CD spectrum of 7

301

showed a negative Cotton effect at 282 nm, indicating that the absolute configuration

302

of 7 was (7R,8S).25,26 Therefore, the structure of 7 was assigned as

303

7R,8S-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-5-(3-methoxyallyl)-7-meth

304

oxycoumaran and it was named hawthornnin G.

305

In its HRESIMS, compound 8 exhibited a pseudomolecular ion peak at m/z

306

445.1458 [M + Na]+ (calcd for C21H26O9Na, 445.1469), which is compatible with the

307

molecular formula C21H26O9. The UV spectrum showed absorbances at λmax 230 and

308

280 nm and the IR spectrum of 8 showed the presence of an aromatic moiety (1604

309

and 1514 cm-1). Inspection of the NMR data (Tables 1 and 2) of 8 showed signals for

310

two substituted aromatic rings, a characteristic doublet at δH 5.56 (1H, d, J = 6.2 Hz,

311

H-7) for dihydrobenzofuran-type lignan, and three OMe groups. A detailed NMR data

312

analysis revealed the similarity between 8 and the known compound bennettin,

313

previously isolated from Flacourtia ramontch,30 suggesting that they shared the same

314

structural skeleton. The trans-relative configuration of C-7 and C-8 was determined

315

by comparison of the coupling constant of H-7 and H-8 (6.2 Hz) with that in the 16


Page 16 of 34

Page 17 of 34


316

literature.25 The absolute configuration at C-7 and C-8 of 8 was assigned to be R and S

317

based on the negative CD Cotton effects at 281 nm.25,26 Furthermore, according to the

318

literature,31 the chemical shifts of C-8′ and C-7′ and the value of ΔδC8′-C7′ were

319

different due to the threo and erythro isomers. The relative configuration of C-7′ and

320

C-8′ was determined to be threo by its 13C NMR data at δC 75.4 (C-7′) and 77.5 (C-8′)

321

and the value of ∆δC8′-C7′ > 1.0 ppm. Consequently, the full structure of 8 was deduced

322

and it was named hawthornnin H.

323

The known compounds from the seeds of Crataegus pinnatifida were identified

324

by comparing their physical and spectroscopic data with values reported in the

325

literature. They are 7R,8S-erythro-dihydroxydehydrodiconiferyl alcohol (9),32

326

7S,8R-erythro-dihydroxydehydrodiconiferyl

327

7R,8S-threo-dihydroxydehydrodiconiferyl

328

7S,8R-threo-dihydroxydehydrodiconiferyl

329

7S,8R-5-methoxydihydrodehydroconiferyl alcohol (14)

330

(15).35

alcohol alcohol alcohol

(12),31 34

7S,8R-ficusal

(10),31 (11),32 (13),33

and 7R,8S-sakuraresinol

331

Various lignans are distributed in significant concentrations in edible plants,

332

especially in flax seed, sesame seed, and other oil seeds.36-38 Most lignans possess a

333

phenolic hydroxyl group in their structures and, therefore, they should exhibit high

334

antioxidant ability.

335

The DPPH and ABTS•+ radical-scavenging assay is usually used to evaluate the

336

antioxidant abilities of extracts or pure compounds.39 The radical scavenging 17



337

activities of all isolates are summarized in table 3. In the DPPH assay, compounds

338

1−6 and 8 showed more potent activity in the DPPH assay, although they are slightly

339

weaker than trolox, and the other compounds only exhibited weak antioxidant activity.

340

In the ABTS assay, most of the isolated compounds exhibited significant antioxidant

341

activity, with compounds 1−6 being the most potent (IC50, 10.6−14.3 µM), while

342

compounds 7-15 exhibited moderate ABTS radical scavenging activities (IC50,

343

18.7−49.8 µM), comparable with that of the standard compound trolox, with an IC50

344

value of 23.7 µM. A comparison of the structures of 1−6 with those of 7−15 indicated

345

that the additional 3-methoxy-4-hydroxy-benzene moiety groups at C-8′ appeared to

346

increase the antioxidant activity of the dihydrobenzofuran neolignans. This fact

347

indicated that the free phenolic hydroxyl was crucial for the antioxidant activity,

348

which was consistent with previous observations.40

349

Pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6, IFN-γ) and NO are the

350

key mediators for the pathogenesis of osteoarthritis, inflammatory bowel diseases,

351

psoriasis, ulcerative colitis and rheumatoid arthritis. Hence, the inhibition of

352

proinflammatory cytokines and NO will be an important strategy for the treatment of

353

these inflammatory conditions.23,41,42 In this investigation, we further evaluated the

354

anti-inflammatory activities of the isolated compounds 1−15 using exposure to

355

LPS-induced NO and TNF-α production in the mouse macrophage RAW264.7 cell

356

line. At a concentration of 100 µM, the MTT assay results showed that 1−15 did not

357

affect cell viability (data not shown). Thus, the effects of compounds 1−15 on 18


Page 18 of 34

Page 19 of 34


358

LPS-induced production of the inflammatory mediators NO and TNF-α in RAW

359

264.7 cells were evaluated at concentrations lower than 100 µM.

360

As shown in tables 4 and 5, compounds 1−7, 13 and 14 showed potent inhibition

361

of NO and TNF-α production. In particular, the activities of 7, 13 and 14 were more

362

potent than that of the positive control. The observed NO and TNF-α inhibitory

363

activities appear to be correlated with their structures. For example, regarding the

364

results for 14 and 15, it appeared that the 4-OH might be important for higher

365

activities. In addition, comparing the structures and inhibitory activities of 7 and 14

366

with those of 8−12, it appeared that the two hydroxyl groups at C-7′, 8′ may cause a

367

reduction in the inhibition of NO and TNF-α production. Interestingly, consideration

368

of the structures of 1-6 vs 8-12 suggested that 8′-OH was replaced by a

369

3-methoxy-4-hydroxy benzoic group resulting in an increase in the NO and TNF-α

370

inhibitory activity of those dihydrobenzofuran neolignans which have the 7, 8-OH

371

moiety in their side chain. A structure-activity relationship study of the related diverse

372

derivatives might be needed to identify the key moiety in dihydrobenzofuran

373

neolignans responsible for inhibiting NO and TNF-α production.

374

In conclusion, 15 dihydrobenzofuran neolignans were isolated from the active

375

fraction of Crataegus pinnatifida seeds and evaluated for inhibition of NO and TNF-α

376

along with antioxidant activities. The results of the present investigations indicate the

377

importance of these neolignans as potential anti-inflammatory and antioxidant agents.

378

From the activity results of the tested compounds, 1-6 showed promising activity 19



379

against NO and TNF-α with less cytotoxicity and considerable antioxidant activity.

380

The results of 7, 13 and 14 as inhibitors of NO and TNF-α are certainly encouraging,

381

but the antioxidant activity of these compounds possibly limits their therapeutic

382

applications. The structure-activity relationship of isolated lignans was also discussed.

383

It is notable that the additional 3-methoxy-4-hydroxy-benzene moiety at position C-8′

384

appeared to increase the antioxidant and anti-inflammatory activities of the

385

dihydrobenzofuran neolignans. The results obtained in our study provide a potential

386

justification for the use of the seeds from Crataegus pinnatifida industrial by-products

387

as a valuable source of raw material for new antioxidant and anti-inflammatory

388

agents.

389

ASSOCIATED CONTENT

390

Supporting Information. UV, CD, IR, NMR, HRESIMS data for compounds

391

1−8. This information is available free of charge via the Internet at http://pubs.acs.org.

392

ACKNOWLEDGEMENT

393

The financial support by the Program for the National Natural Science Foundation

394

of China (81302661) and the Scientific Research Starting Foundation (20121106) for

395

Doctors of Liaoning province of P.R. China is gratefully acknowledged.

396

ABBREVIATIONS USED

397

NMR, nuclear magnetic resonance spectrometry; HPLC, high-performance 20


Page 20 of 34

Page 21 of 34


398

liquid chromatography; HSQC, heteronuclear single quantum coherence; HMBC,

399

heteronuclear multiple bond correlation; NOESY, nuclear overhaser effect

400

spectroscopy; ESI-MS, electrospray ionization mass spectrometry; UV, ultraviolet

401

spectrometry; CD, electronic circular dichroism; IR, infared absorption spectrum.

402

21



Page 22 of 34

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(38) Peñalvo, J. L.; Adlercreutz, H.; Uehara, M.; Ristimaki, A.; Watanabe, S. Lignan content of selected foods from Japan. J. Agric. Food Chem. 2008, 56, 401-409. (39) Chung, Y. M.; Wang, H. C.; El-Shazly, M.; Leu, Y. L.; Cheng, M. C.; Lee. C, L.; Chang, F. R.; Wu, Y. C. Antioxidant and tyrosinase inhibitory constituents from a desugared sugar cane extract, a byproduct of sugar production. J. Agric. Food Chem. 2011, 59, 9219-9225. (40) Hamerski, L.; Bomm, M. D.; Silva, D. H. S.; Young, M. C. M.; Furlan, M.; Eberlin, M. N.; Castro-Gamboa, I.; Cavalheiro, A. J.; Bolzani, V. D. S. Phenylpropanoid glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae). Phytochemistry 2005, 66, 1927-1932. (41) Pu, W.; Lin, Y.; Zhang, J.; Wang, F.; Wang, C.; Zhang, G. 3-Arylcoumarins: Synthesis and potent anti-inflammatory activity. Bioorg. Med. Chem. Lett. 2014, 24, 5432-5434. (42) Patel, N. K.; Bairwa, K.; Gangwal, R.; Jaiswal, G.; Jachak, S. M.; Sangamwar, A. T.; Bhutani, K. K. 2′-Hydroxy flavanone derivatives as an inhibitors of pro-inflammatory mediators: Experimental and molecular docking studies. Bioorg. Med. Chem. Lett. 2015, 25, 1952-1955.

26


Page 26 of 34

Page 27 of 34


FIGURE CAPTIONS Fig. 1 Structures of compounds 1-15.

27



Page 28 of 34

Fig. 2 The Key HMBC correlations of 1 and 5 OH

OCH3 OH

OCH3 OH

OH HO

HO OCH3

HO

O

HO O

O H3CO

OCH3

H3CO

1

OCH3 5

28


Page 29 of 34


Table 1 1H NMR data for compounds 1-8 (600 MHz) H

1a

2a

2

6.89 (d, 1.6)

6.91 (br.s)

5

6.75 (d, 8.1)

6.78 (br.s)

6.83 (dd, 8.2,

6.83 (dd, 8.1,

6.77 (dd, 8.1, 6

3a

4a

5a

6a

7a

8b

6.95 (d, 1.8)

6.96 (d, 1.8)

6.79 (br.s)

6.79 (d, 1.6)

6.91 (br.s)

6.69 (s)

6.78 (d, 8.2)

6.78 (d, 8.1)

6.66 (br.s)

6.65 (d, 8.1)

6.76 (br.s)

―

6.76 (br.s)

6.69 (s)

5.38 (d, 5.5)

5.47 (d, 6.6)

5.56 (d, 6.2)

3.31 (m)

3.44 (m)

3.52 (m)

10.9, 5.2)

3.62 (m),

3.88 (m),

3.44 (dd,

3.70 (m)

3.79 (m)

6.97 (br.s)

6.97 (br.s)

6.78 (br.s) 1.6)

7 8

5.49 (d, 5.5)

5.48 (d, 6.0)

3.40 (m)

3.41 (m)

3.67 (dd,

6.67 (dd, 8.1, 6.66 (br.s)

1.8)

1.7)

5.53 (d, 5.8)

5.53 (d, 6.3)

5.36 (d, 6.1) 3.32 (m)

3.46 (m)

3.47 (m)

3.67 (dd,

3.69 (dd,

11.5, 7.7)

10.7, 7.7)

1.6)

3.56 (dd, 3.68 (dd, 9.2,

11.0, 5.1)

3.79 (m)

3.3)

9 3.48 (dd,

3.70 (m)

3.76 (dd,

3.85 (dd, 3.58 (m)

11.0, 8.3) 2′

6.51 (br.s)

6.51 (br.s)

11.5, 6.4)

10.7, 2.9)

6.50 (br.s)

6.49 (br.s)

10.9, 8.3) 6.41 (br.s)

6.43 (br.s)

6′

6.54 (br.s)

6.63 (br.s)

6.61 (br.s)

6.68 (br.s)

6.52 (br.s)

6.44 (br.s)

6.97 (br.s)

6.92 (br.s)

7′

4.32 (d, 8.6)

4.35 (d, 8.3)

4.55 (d, 5.2)

4.53 (d, 5.5)

4.33 (d, 8.4)

4.31 (d, 8.6)

6.52 (d, 15.9)

4.59 (d, 6.0)

8′

2.96 (m)

3.03 (m)

286 (m)

2.89 (m)

2.90 (m)

2.85 (m)

6.18 (dt, 3.71 (m) 15.9, 6.0) 3.92 (dd,

3.92 (dd,

3.89 (dd,

3.99 (dd,

4.00 (dd,

10.7, 6.8)

10.9, 5.8)

10.9, 5.2)

3.92 (dd, 10.9, 7.1)

11.0, 6.8)

9′

10.7, 7.2) 4.07 (dd,

4.07 (dd,

10.9, 5.0)

11.0, 5.3)

2′′

6.43 (d, 1.8)

6.49 (br.s)

5′′

6.61 (d, 8.1)

6.63 (d, 7.3)

3.52 (m), 4.00 (d, 6.0)

4.76 (dd,

3.81 (dd,

3.82 (dd,

10.7, 4.3)

10.9, 7.2)

10.9, 8.3)

6.60 (d, 1.7)

6.63 (d, 1.6)

6.39 (d, 1.1)

6.35 (d, 1.8)

6.69 (d, 8.0)

6.69 (d, 8.2)

6.51 (d, 8.2)

6.52 (d, 8.1)

6.58 (d, 8.1,

6.60 (d, 8.2,

6.42 (d, 8.0,

6.41 (d, 8.1,

1.7)

1.6)

1.1)

1.8)

3.83 (3H, s)

3.83 (3H, s)

3.69 (3H, s)

3.68 (3H, s)

3.41 (m)

3.76 (m)

6.50 (dd, 8.1, 6′′

6.52 (d, 7.3) 1.8)

3-OCH3

3.79 (3H, s)

3.81 (3H, s)

3.80 (3H, s)

5-OCH3

3.83 (3H, s) 3.83 (3H, s)

3′-OCH3

3.72 (3H, s)

3.72 (3H, s)

3.73 (3H, s)

3.76 (3H, s)

3.60 (3H, s)

3.62 (3H, s)

3′′-OCH3

3.64 (3H, s)

3.70 (3H, s)

3.72 (3H, s)

3.71 (3H, s)

3.59 (3H, s)

3.55 (3H, s)

7′-OCH3

3.24 (3H, s)

3.24 (3H, s)

3.20 (3H, s)

3.20 (3H, s)

9′-OCH3

3.74 (3H, s)

3.91 (3H, s)

3.26 (3H, s)

7′-OCH2CH3

3.30 (2H, m);

3.30 (2H, m);

1.08 (3H, t,

1.07 (3H, t,

7.0)

7.0)

Coupling constants (J) in Hz are given in parentheses; Chemical shift values are expressed in ppm. a

Measured in CD3OD; b Measured in DMSO-d6.

29



Page 30 of 34

Table 2. 13C NMR data (150 MHz) of compounds 1-8. No.

1a

2a

3a

4a

5a

6a

7a

8b

1

134.8

134.6

134.8

134.7

134.6

134.8

132.2

136.4

2

110.4

110.6

110.5

110.6

110.6

110.4

110.5

104.2

3

149.0

149.1

149.1

149.1

149.1

149.0

147.4

149.4

4

147.4

147.5

147.5

147.5

147.5

147.4

146.4

133.9

5

116.1

116.2

116.1

116.2

116.2

116.1

115.3

149.4

6

119.5

119.7

119.5

119.7

119.7

119.4

118.5

104.2

7

89.2

89.3

89.2

89.2

89.3

89.2

87.4

89.3

8

55.3

55.1

55.3

55.3

55.1

55.3

52.9

55.5

9

65.4

65.1

65.3

65.0

65.1

65.3

62.9

64.9

1′

134.9

134.8

135.4

135.3

135.5

135.6

130.0

137.0

2′

113.3

113.3

112.8

112.8

113.2

113.1

110.3

116.6

3′

144.9

144.9

145.1

145.1

144.9

144.9

143.7

145.3

4′

148.5

148.6

148.6

148.7

148.5

148.4

147.5

148.9

5′

129.2

129.4

129.3

129.5

129.4

129.1

129.5

129.7

6′

117.5

117.5

117.1

117.2

117.3

117.3

115.2

112.7

7′

87.5

87.5

84.8

85.0

85.9

85.9

131.9

75.4

8′

56.7

56.6

56.5

56.5

56.2

56.3

123.6

77.5

9′

64.9

64.9

64.3

64.3

65.0

65.0

72.3

64.3

1′′

132.6

132.6

132.1

132.2

132.7

132.7

2′′

114.4

114.4

114.7

114.8

114.4

114.4

3′′

148.3

148.3

148.3

148.3

148.8

148.4

4′′

146.0

146.1

146.1

146.2

146.1

146.0

5′′

115.7

115.7

115.5

115.6

115.7

115.7

6′′

122.5

122.6

123.3

123.2

122.5

122.5

3-OCH3

56.3

56.4

56.5

56.5

56.6

56.7

55.6

56.8

5-OCH3

56.8

3′-OCH3

56.3

56.4

56.4

56.4

56.4

56.4

3′′-OCH3

56.3

56.1

56.4

56.4

56.4

56.3

7′-OCH3

57.0

56.9

57.2

57.2

9′-OCH3

57.1

7′-OCH2CH3 a

55.6

65.2

65.5

15.7

15.7

measured in CD3OD; b measured in DMSO-d6.

30


56.8

Page 31 of 34


Table 3. Antioxidant activities of compounds 1-15 Compound

DPPH (IC50, µM)

ABTS (IC50, µM)

1

82.1 ± 1.8

11.7 ± 0.7

2

79.2 ± 1.8

10.6 ± 1.1

3

75.1 ± 1.2

12.6 ± 0.6

4

72.0 ± 0.9

12.0 ± 1.2

5

89.0 ± 1.0

13.6 ± 0.9

6

89.5 ± 2.6

14.3 ± 1.2

7

323.1 ± 1.4

35.51 ± 2.1

8

71.1 ± 2.5

30.7 ± 1.8

9

279.7 ± 5.1

23.4 ± 0.9

10

298.5 ± 3.3

24.5 ± 0.8

11

309.9 ± 2.5

29.6 ± 0.9

12

299.5 ± 3.00

31.3 ± 1.3

13

202.2 ± 1.3

49.8 ± 1.4

14

252.9 ± 2.4

18.7 ± 0.9

15

242.0 ± 2.1

24.8 ± 1.1

Trolox

31.4 ± 0.2

23.7 ± 0.3

IC50 values represent the means ± S.E.M. of three parallel measurements.

31



Page 32 of 34

Table 4. Effects of compounds 1-15 on nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells Compound 1 2

Inhibition (%) 0 µM

1 µM

10 µM *

0.0 ± 4.7

10.4 ± 3.2

0.0 ± 4.2

*

8.3 ± 4.6

*

IC50 50 µM

*

13.3 ± 4.7

**

21.2 ± 2.0

*

57.1 ± 2.1

**

49.8 ± 2.4

**

30.5

**

27.3

**

82.0 ± 1.5 85.1 ± 1.1

0.0 ± 6.8

13.6 ± 2.2

12.1 ± 3.1

37.8 ± 1.6

87.1 ± 3.9

35.3

4

0.0 ± 6.2

8.2 ± 1.7

12.9 ± 1.2*

65.6 ± 1.8**

80.2 ± 2.5**

29.1

3.1 ± 4.4

**

**

**

26.0

**

29.4

**

14.1

**

86.8

**

6 7 8

0.0 ± 3.9 0.0 ± 3.2 0.0 ± 4.4 0.0 ± 5.7

14.8 ± 2.0

2.6 ± 3.1

11.3 ± 2.4

** **

6.7 ± 3.3

64.8 ± 2.2

*

3.7 ± 3.5

10.2 ± 2.9

**

51.1 ± 2.7

**

61.7 ± 2.6

**

67.1 ± 3.5

**

38.0 ± 2.1

**

92.6 ± 3.5 87.9 ± 1.4 82.0 ± 2.4 59.8 ± 1.3

9

0.0 ± 5.9

6.9 ± 2.8

20.8 ± 3.6

39.4 ± 1.2

47.2 ± 2.1

> 100

10

0.0 ± 4.6

9.5 ± 3.8*

16.4 ± 1.3**

29.8 ± 3.3*

42.1 ± 2.2**

> 100

*

**

**

> 100

**

> 100

**

11.3

**

8.8

**

11 12 13 14

0.0 ± 4.0 0.0 ± 5.7 0.0 ± 4.4 0.0 ± 6.4

*

10.1 ± 4.9

10.9 ± 3.1

**

4.5 ± 3.2

19.1 ± 3.6 *

13.1 ± 5.7

**

21.8 ± 2.1

**

15

0.0 ± 2.0

9.3 ± 1.2

Mino

0.0 ± 3.2

15.6 ± 1.6**

**

49.2 ± 2.5

**

48.3 ± 1.9

**

31.6 ± 3.7

**

22.1 ± 2.4

**

77.2 ± 3.3

**

75.5 ± 1.5

**

44.9 ± 1.0 40.6 ± 2.5

82.5 ± 3.2 82.1 ± 1.2

16.5 ± 2.2

24.2 ± 3.1

34.2 ± 1.4

> 100

22.7 ± 2.1**

51.8 ± 2.8**

59.7 ± 1.9**

55.1

Each value represents the means ± S.E.M. of three parallel measurements. Significantly different from the control. p < 0.05.

**

**

3 5

*

(µM)

100 µM **

p < 0.01.

32


Page 33 of 34


Table 5. Effects of compounds 1-15 on tumor necrosis factor α (TNF-α) production in LPS-stimulated RAW 264.7 cells Compound 1 2

Inhibition (%) 0 µM 0.0 ± 3.7 0.0 ± 4.3

1 µM 7.1 ± 2.6

10 µM *

10.5 ± 2.0

*

IC50 50 µM

23.5 ± 1.0

**

30.2 ± 2.3

** **

(µM)

100 µM

46.5 ± 1.4

**

49.1 ± 3.1

**

44.7 ± 1.8

**

55.3 ± 3.4

**

67.4

59.5 ± 2.1

**

50.2

52.4 ± 1.0

**

63.1

3

0.0 ± 3.5

4.1 ± 2.5

32.1 ± 2.7

4

0.0 ± 3.2

6.9 ± 3.0*

33.5 ± 1.5**

43.6 ± 3.3**

61.5 ± 2.7**

50.8

5.3 ± 2.2

23.1 ± 1.8

**

41.2 ± 1.3

**

51.1 ± 2.5

**

85.0

21.5 ± 2.9

**

48.0 ± 1.7

**

57.0 ± 1.9

**

71.8

31.0 ± 3.2

**

55.3 ± 2.2

**

69.5 ± 2.0

**

31.4

19.2 ± 2.6

**

29.5 ± 3.9

**

35.1 ± 3.7

**

> 100

24.3 ± 1.8

**

28.6 ± 3.2

**

> 100

5 6 7 8

0.0 ± 5.2 0.0 ± 3.2 0.0 ± 5.7 0.0 ± 4.0

15.0 ± 2.3

**

11.3 ± 3.0

*

3.2 ± 2.0

*

9

0.0 ± 5.0

2.4 ± 3.5

11.9 ± 3.4

10

0.0 ± 4.2

5.2 ± 4.7

17.2 ± 2.2**

21.8 ± 2.3**

23.1 ± 3.4**

> 100

0.0 ± 2.4

*

**

25.6 ± 2.0

**

31.2 ± 2.8

**

> 100

27.2 ± 3.5

**

34.0 ± 1.3

**

> 100

62.1 ± 3.2

**

69.8 ± 1.6

**

15.2

51.2 ± 1.4

**

63.8 ± 2.7

**

41.4

27.3 ± 1.9

**

42.1 ± 2.2

**

> 100

11 12 13 14

0.0 ± 5.7 0.0 ± 2.8 0.0 ± 3.7

7.2 ± 4.3

3.1 ± 4.2 21.0 ± 2.4

**

14.6 ± 3.2

**

15

0.0 ± 4.5

7.5 ± 4.2

Silybin

0.0 ± 3.4

10.3 ± 2.5**

19.1 ± 3.5

14.2 ± 2.6

*

49.2 ± 3.7

**

29.3 ± 2.5

**

16.5 ± 1.6

**

27.0 ± 3.8**

47.0 ± 3.1**

Each value represents the means ± S.E.M. of three parallel measurements. Significantly different from the control. * p < 0.05. ** p < 0.01.

403

33


53.8 ± 3.7**

69.2


TOC Graphic

34


Page 34 of 34

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