Flavanones and Chromones from Salicornia herbacea Mitigate Septic

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Flavanones and Chromones from Salicornia herbacea Mitigate Septic Lethality via Restoring Vascular Barrier Integrity Nguyen Quoc Tuan, Wonhwa Lee, Joonseok Oh, Roshan Rajan Kulkarni, Charlotte Gény, Byeongjin Jung, Hyejin Kang, Jong-Sup Bae, and MinKyun Na J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04069 • Publication Date (Web): 31 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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Journal of Agricultural and Food Chemistry

Flavanones and Chromones from Salicornia herbacea Mitigate Septic Lethality via Restoring Vascular Barrier Integrity Nguyen Quoc Tuan,†,‡,# Wonhwa Lee,§,⊥,# Joonseok Oh,║ Roshan Rajan Kulkarni,† Charlotte Gény,║ Byeongjin Jung,§ Hyejin Kang,§ Jong-Sup Bae,*,§ MinKyun Na*,† †

College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea.

‡

Phutho College of Pharmacy, Viettri City, Phutho Province, Vietnam

§

College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, ⊥BK21 Plus

KNU Biomedical Convergence Program, Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu 41566 and 41944, Republic of Korea ║

Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute

of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, United States

1 ACS Paragon Plus Environment


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1

ABSTRACT: Salicornia herbacea is an annual halophytic glasswort that has been employed

2

as a culinary vegetable, salad, and traditional medicinal resource. Chemical investigation of

3

the aerial parts of S. herbacea led to the isolation of two new (1, 2) and known (3) flavanone

4

as well as a new nature-derived (4) and two known chromone derivatives (5, 6). These

5

purified compounds were evaluated for their suppressive potentials against the release of high

6

mobility group box 1 protein (HMGB1) which has captured the attention as a viable target for

7

alleviating serious septic manifestations or septicemia. The phenolic compounds improved

8

the survival rates of cecal ligation and puncture operation (CLP) in murine models,

9

simulating severe septic shock and its related complications, to 40–60%. These results

10

collectively validate that flavanone- and chromone-based secondary metabolites may serve as

11

prospective prodrugs or food additives that may be commercialized for the control of septic

12

complications and lethality.

13 14

KEYWORDS: Salicornia herbacea, flavanones, chromones, severe sepsis. endothelial

15

barrier integrity, high mobility group box 1 protein


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INTRODUCTION

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Sepsis is a systemic and excessive inflammatory response to a local infection, possibly

18

proceeding to severe sepsis and septic complications such as multiple organ failure and

19

hypotension.1 Hospitalization rates for septicemia have doubled from 2000 through 2008 in

20

the U.S.2 and the case fatality rate has ranged 20–30% during the past two decades.3

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Conventional anti-inflammatory treatments and advanced life-support have not mitigated

22

septicemic manifestations thus far,3 which prompts the rigorous exploration of a sensible

23

strategy for the control of this serious disease.

24

reinforcement of the barrier integrity of endothelial cells (ECs) is a viable mode of action for

25

sepsis treatment.4,5 Among numerous approaches to enhance the cellular integrity, inhibition

26

of high mobility group box 1 (HMGB1) protein has emerged as a practical protocol because

27

of the predominant role of HMGB1 in disturbing the vascular barrier integrity, and thereby

28

causing septicemia and its related serious complications.4-6

29

Salicornia herbacea L. (Chenopodiaceae), also known as glasswort, sea antler, or sea

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cordyceps, is an annual halophytic herb mainly inhabiting muddy seashores, salt marshes,

31

and salt lakes.7-9 This plant has been employed not only as a seasoned vegetable and

32

fermented food in coastal areas but as traditional medicine to treat constipation, obesity,

33

diabetes, nephropathy, hepatitis and diarrhea.10,11 Chemical investigations identified various

34

bioactive scaffolds such as phytosterols, phenolic acids, flavonoids, caffeoyl quinic acids,

35

procatechuic acids, and triterpenoid saponins.10,12,13 Biological activities of some of these

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diverse classes of compounds have been validated as well. Flavonoids from this salt-tolerant

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plant have displayed anti-adipogenic potential8 and inhibitory activity on matrix

38

metalloproteinase associated with pro-inflammatory pathways.14 Our previous study of S.

39

herbacea also validated that quinic acid derivatives significantly alleviated HMGB1-induced

40

inflammatory stress in vascular barriers, which leads to improving survival rates of severe

41

septic in vivo models.13

Recent studies have verified that



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In continuing efforts to search for natural product-derived prodrugs or food components that

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ameliorate severe sepsis and septic manifestations,13,15,16 the current study delineates the

44

isolation and structure elucidation of two new and one known flavanones (1‒3), and a new

45

nature-derived (4) and two known chromone derivatives (5, 6) from the EtOAc extract of S.

46

herbaceae exhibiting potent anti-HMGB1 activity in our previous study.13 The isolated

47

compounds were evaluated for their in vitro and in vivo inhibitory potential against vascular

48

inflammatory stimulations. Detailed biological evaluation and mechanistic studies were also

49

performed using relevant cellular pathways and animal models to ultimately verify whether

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the phytochemicals may be developed into a prospective food additive or drug lead to control

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serious septic complications.

52 53

MATERIALS AND METHODS

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Reagents and Chemicals. Bacterial lipopolysaccharide (LPS) (serotype: 0111:B4,

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L5293, used at 100 ng/ mL), Evans blue, and crystal violet were obtained from Sigma-

56

Aldrich (St. Louis, MO, USA), and Vybrant DiD (used at 5 µM) was obtained from

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Invitrogen (Carlsbad, CA, USA). Human recombinant HMGB1 was purchased from Abnova

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(Taipei City, Taiwan). Acetic acid was purchased from Samchun pure chemical Co., Ltd

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(Hwaseong, Korea), and HPLC grade MeOH was purchased from Tedia (Fairfield, OH,

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USA). Bulk MeOH, hexanes and acetone used for extraction and isolation were purchased

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from SK chemicals (Seongnam, Korea).

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Plant Material. The aerial parts of S. herbacea were purchased from a farm (Shinan,

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Korea) in 2012 and authenticated by Prof. Hye Gwang Jeong and MinKyun Na (College of

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Pharmacy, Chungnam National University). A voucher specimen (CNU02013_01) was

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deposited at the Pharmacognosy laboratory of the College of Pharmacy, Chungnam National

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University (Daejeon, Korea).


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Extraction and Isolation of Active Metabolites. The details of the extraction

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process are elaborated in our previous study.13 The EtOAc-soluble fraction (45.7 g) was

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subjected to silica vacuum liquid chromatography (VLC) (length 25 × i.d. 18 cm, 100―200

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µm) and eluted with hexanes/acetone (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 30:70, 10:90,

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0:100; each step 2 L; a final washing with 6 L of 100 % MeOH) to acquire nine fractions E1

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(1.4 mg), E2 (9.3 mg), E2 (1.40 g), E4 (1.30 g), E5 (2.77 g), E6 (2.80 g), E7 (5.40 g), E8

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(9.53 g), E9 (18.03 g). Among these VLC fractions, the fraction E5 (2.77 g) was further

74

chromatographed on reverse phase MPLC (Biotage KP-C18-HS, 400 g) eluting with isocratic

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60 % of MeOH in H2O to give seven sub-fractions E5-1 (458 mg), E5-2 (275 mg), E5-3 (280

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mg), E5-4 (220 mg), E5-5 (241 mg), E5-6 (620 mg), and E5-7 (600 mg). The fraction E5-3

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was purified with HPLC (Phenomenex Luna C18 250 × 21.20 mm, 5 µm), eluting with a

78

gradient mixture of MeOH/H2O (0-10 min: 50% MeOH; 60 min: 80% MeOH; 70 min: 100%

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MeOH) to furnish compound 1 (tR : 26 min, 7.6 mg) and six sub-fractions E5-3-1 (32.3 mg),

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E5-3-2 (24.0 mg), E5-3-3 (39 mg), E5-3-4 (33.6 mg), E5-3-5 (15.5 mg), and E5-3-6 (4.2 mg).

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The fraction E5-3-2 was further separated with HPLC, eluting by using a program linear

82

gradient A-B, 0-10 min, 50% A, 60 min, 68% A, 65 min, 100% A to isolate compound 2 (tR :

83

52 min, 5.2 mg). The fraction E5-4 was further fractionated using HPLC, eluting with a

84

gradient mixture of MeOH/H2O (0-10 min: 50% MeOH, 50 min: 80% MeOH, 70 min: 100%

85

MeOH) to afford eight sub-fractions E5-4-1 (17.7 mg), E5-4-2 (3.2 mg), E5-4-3 (3.6 mg),

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E5-4-4 (8.0 mg), E5-4-5 (10.3 mg), E5-4-6 (58.4 mg), E5-4-7 (9.0 mg), and E5-4-8 (21.9 mg).

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Compound 3 (tR : 32 min, 5.7 mg) was acquired from one of those sub-fractions E5-4-4 with

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HPLC application eluting with a linear gradient mixture of MeOH/H2O (0-10 min: 50%

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MeOH, 50 min: 80% MeOH, 60 min: 100% MeOH. Fraction E6 was chromatographed over

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reverse phase MPLC (Biotage KP-C18-HS, 120g) with elution of a gradient mixture of

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MeOH/H2O to generate nine fractions E6-1 (121.4 mg), E6-2 (739 mg), E6-3 (175 mg), E6-4

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(312.8 mg), E6-5 (258.7 mg), E6-6 (48.7 mg), E6-7 (90 mg), E6-8 (71.8) mg, and E6-9 (803 5 ACS Paragon Plus Environment


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mg). Further HPLC separation was performed on the fraction E6-2 utilizing the identical

94

gradient method as for the purification of E5-4-4 to afford 14 sub-fractions E6-2-1 (3.1 mg),

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E6-2-2 (0.5 mg), E6-2-3 (0.4 mg), E6-2-4 (40.5 mg) E6-2-5 (8.7 mg), E6-2-6 (16.1 mg), E6-

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2-7 (16.1 mg), E6-2-8 (31.1 mg), E6-2-9 (13.3 mg), E6-2-10 (16.7 mg), E6-2-11 (26.1 mg),

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E6-2-12 (11.4 mg), E6-2-13 (0.6 mg), and E6-2-14 (165.5 mg). The fraction E6-2-8 was

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HPLC-purified using a linear gradient of MeOH/H2O (0-10 min: 15% MeOH, 60 min: 30%

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MeOH, 75 min: 100% MeOH) to isolate compound 5 (tR: 18 min, 3.5 mg). Compound 4 (3.0

100

mg) was re-crystallized from the fraction E6-2-10 in MeOH. Fraction E6-4 (312.8 mg) was

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applied on semi-preparative HPLC (Phenomenex Luna C18 250 × 10 mm, 5 µm) eluting with

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MeOH/H2O (0-10 min: 25% MeOH, 20 min, 30% MeOH, 70 min, 40% MeOH, 90 min: 80%

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MeOH, 110 min: 100% MeOH) to furnish compound 6 (tR : 65 min, 3.5 mg).

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Cell Culture. Primary human umbilical vein endothelial cells (HUVECs) were obtained

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from Cambrex Bio Science (Charles City, IA, USA) and maintained as described

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previously.14 Briefly, cells were cultured to confluence at 37 ˚C and 5% CO2 in an EBM-2

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basal medium with growth supplements (Cambrex Bio Science, East Rutherford, NJ, USA).

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THP-1 cells were maintained at a density of 2 × 105 to 1 × 106 cells/mL in RPMI 1640 with

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L-glutamine

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mercaptoethanol (55 µM), and penicillin G and streptomycin as antibiotics.

and 10% heat-inactivated fetal bovine serum (FBS) supplemented with 2-

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Animals and Husbandry. Male C57BL/6 mice (6–7 weeks old, weighing 18–20 g)

112

were purchased from Orient Bio Co. (Seongnam, Korea) and used after a 12-day

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acclimatization period. Animals were housed five per polycarbonate cage under controlled

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temperature (20–25 ˚C) and humidity (40–45 %) and a 12:12 h light : dark cycle. A normal

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rodent pellet diet and water ad libitum were provided for animals during acclimatization. All

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animals were treated in accordance with the ‘Guidelines for the Care and Use of Laboratory

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Animals’ issued by Kyungpook National University (IRB No. KNU 2012-13).


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Competitive ELISA for HMGB1. Concentration of HMGB1 was determined using a

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competitive enzyme-linked immunosorbent assay (ELISA) method with reference to a

120

previous study.4 Briefly, HUVEC monolayers were treated with LPS (100 ng/mL) for 16 h

121

followed by treatment of the isolated compounds for 6 h. Cell culture media were collected

122

for the determination of an HMGB1 concentration. For ELISA, 96-well flat plastic microtiter

123

plates (Corning, NY, USA) were coated with HMGB1 in 20 mM carbonate–bicarbonate

124

buffer (pH 9.6) containing 0.02% sodium azide overnight at 4 ˚C. Plates were rinsed three

125

times in PBS-0.05% Tween 20 (PBS-T) and kept at 4 ˚C. Lyophilized culture media were

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pre-incubated with anti-HMGB1 antibodies (Abnova, Taipei City, Taiwan; diluted 1:1000 in

127

PBS-T) in 96-well plastic round microtiter plates for 90 min at 37 ˚C. Pre-incubated samples

128

were transferred to pre-coated plates and incubated for 30 min at room temperature. Plates

129

were rinsed three times in PBS-T, incubated for 90 min with peroxidase-conjugated anti-

130

rabbit IgG antibodies (Amersham Pharmacia Biotech, Piscataway, NJ, USA; diluted 1:2000

131

in PBS-T), re-rinsed three times with PBS-T, and re-incubated for 60 min in the dark with

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200 µL substrate solution (100 µg/mL o-phenylenediamine and 0.003 % H2O2). After

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quenching the reaction with 50 µL of 8 N H2SO4, absorbance was read at 490 nm.

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Cell Viability Assay (MTT Assay). Cells were grown in 96-well plates at a density of 5

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× 103 cells/well. After 24 h, cells were washed with fresh media, followed by treatment with

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the purified compounds. After a 48 h incubation period, cells were washed, and 100 µl of 3-

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(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1 mg/mL) was added,

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followed by incubation for 4 h. DMSO (150 µl) was added to solubilize the formazan salt

139

formed during the reaction and the generated amount of formazan salt was determined by

140

measuring the OD at 540 nm utilizing a microplate reader (Tecan Austria GmbH, Grödig,

141

Austria).

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Expression of CAMs and Receptor Expression. Expression of VCAM-1,

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intercellular adhesion molecule-1 ICAM-1, and E-selectin on HUVECs was determined by a 7 ACS Paragon Plus Environment


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whole-cell ELISA technique as delineated previously.15,16 HUVEC monolayers were treated

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with HMGB1 (1 µg/mL) for 16 h, followed by treatment with the isolated compounds from S.

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herbacea for 6 h. Media were removed, and cells were washed with PBS and fixed with 50

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µL of 1% paraformaldehyde for 15 min at room temperature. After washing, 100 µL of

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mouse anti-human monoclonal antibodies for each adhesion factor, i.e., VCAM-1, ICAM-1,

149

and E-selectin (Temecula, CA, USA; 1:50 each) were applied. After 1 h (37 ˚C, 5% CO2),

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cells were washed three times, followed by addition of 100 µL of 1:2000 peroxidase-

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conjugated anti-mouse IgG antibodies (Sigma-Aldrich, Saint Louis, MO, USA) for 1 h. Cells

152

were re-washed three times and developed using o-phenylenediamine as a substrate (Sigma-

153

Aldrich, St. Louis, MO, USA). Colorimetric analysis was performed by measuring

154

absorbance at 490 nm. All measurements were performed in triplicate wells. The identical

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experimental procedures were utilized for assessing cell surface expression of TLR2, TLR4,

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and RAGE receptors using specific antibodies for each receptor, i.e., A-9, H- 80, and A-9,

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respectively (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

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Permeability Assay In Vitro. Endothelial cell permeability in response to increasing

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concentrations of compounds 1–6 was quantified by spectrophotometric measurement of the

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flux of Evans blue-bound albumin across cell monolayers and the measurement was achieved

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using a two-compartment chamber model.17 HUVECs were plated (5 × 104/well) in 3-µm

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pore size, 12-mm diameter transwells for three days. Confluent monolayers of HUVECs were

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treated with HMGB1 (1 µg/mL) for 16 h or LPS (100 ng/mL) for 4 h followed by treatment

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with the purified compounds for 6 h. Transwell inserts were washed with PBS (pH 7.4) and

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0.5 mL of Evans blue (0.67 mg/mL) diluted in growth medium containing 4% BSA was

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added. Fresh growth medium was added to the lower chamber, and the medium in the upper

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chamber was replaced with Evans blue/BSA. Optical density was measured at 650 nm in the

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lower chamber after 10 min.


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Migration Assay In Vitro. Migration assays were performed in transwell plates with a

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diameter of 6.5 mm with filters (pore size of 8 µm). HUVECs (6 × 104) were cultured for

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three days to obtain confluent endothelial monolayers. Before THP-1 cells were added to the

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upper compartment, cell monolayers were treated with HMGB1 (1 µg/mL) for 16 h followed

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by treatment with the phenolic compounds for 6 h. Cells in the upper chamber of the filter

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were aspirated and non-migrating cells on top of the filter were removed with a cotton swab.

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Monocytes on the lower side of the filter were fixed with 8% glutaraldehyde and stained with

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0.25% crystal violet in 20% MeOH (w/v). Each experiment was repeated in duplicate wells

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and within each well, nine randomly selected high power microscopic fields (200 ×) were

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counted and expressed as a migration index.

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In Vivo Permeability and Leukocyte Migration Assay. For in vivo migration

180

study, male mice were anesthetized with 2% isoflurane (Forane, JW pharmaceutical, Seoul,

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Korea) in oxygen delivered via the rodent gas anesthesia machine RC2 (VetEquip,

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Pleasanton, CA, USA), initially via a breathing chamber followed by a facemask. They were

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allowed to breath spontaneously during the procedure. Mice were intravenously administered

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with HMGB1 (2 µg/mouse). After 6h, murine models were intravenous treatment with 1 (5.7

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µg/mouse), 2 (6.0 µg/mouse), 3 (6.0 µg/mouse), 4 (4.4 µg/mouse), 5 (3.6 µg/mouse), and 6

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(4.1 µg/mouse). Mice were sacrificed 2 h later and peritoneal exudates were collected by

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washing cavities with 5 mL of normal saline and centrifuging at 200 g for 10 min.

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Supernatant absorbance was read at 650 nm. Vascular permeability was expressed as µg of

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dye/mouse that leaked into the peritoneal cavity, and concentrations were determined

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utilizing a standard curve with reference to previously validated procedures.18,19 For

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assessment of leukocyte migration, mice were stressed with HMGB1 (2 µg/mouse,

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intravenous administration) and after 6h treated intravenously with the aforementioned

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concentrations of compounds 1–6. Mice were sacrificed after 2 h and peritoneal cavities were

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washed with 5 mL of normal saline. Samples (20 µL) of obtained peritoneal fluids were 9 ACS Paragon Plus Environment


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mixed with 0.38 mL of Turk’s solution (0.01% crystal violet in 3% acetic acid) and numbers

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of leukocytes were counted under a light microscope.

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Cell–Cell Adhesion Assay. Adherence of monocytes to endothelial cells was assessed

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using fluorescent labeling of monocytes as previously described.12,14,15 Briefly, THP-1 cells

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(1.5 × 106/mL, 200 µL/well) were labeled with Vybrant DiD dye and added to HUVECs

200

stressed with vascular insults. HUVEC monolayers were treated with HMGB1 (1 µg/mL) for

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16 h followed by treatment with tested compounds for 6 h. After THP-1 incubation for 1 h at

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37 °C, non-adherent cells were eliminated by washing four times with pre-warmed RPMI and

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the fluorescent signals of adherent cells were measured. Adherent cells were analyzed

204

utilizing fluorescence microscopy at × 400 (Leica microsystems, Wetzlar, Germany).

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ELISA for Phosphorylated Pro-Inflammatory Factors. Expression of total and

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phosphorylated p38 in whole cell lysates was quantified using a commercially available

207

ELISA kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s

208

instructions. Total and phosphorylated nuclear factor- NF)-κB p65 (Cell Signaling

209

Technology, Danvers, MA, USA) or total and phosphorylated ERK1/2 (R&D Systems,

210

Minneapolis, MN, USA) activities were determined employing the respective ELISA kits.

211

Total NF-κB p65, total and phosphorylated ERK1/2 were measured in whole cell lysates and

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phosphorylated NF-κB p65 was measured in nuclear lysates. The expression of interleukin-

213

(IL)-1β and tumor necrosis factor (TNF)-α in cell culture supernatants was also determined

214

using respective ELISA kits (R&D Systems, Minneapolis, MN, USA).

215

Immunofluorescence (IF) Staining. HUVECs were cultured to confluence on glass

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cover slips coated with 0.05% Poly-L-lysine in complete media containing 10% FBS and

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maintained for 48 h. Cells were stressed with HMGB1 (1 µg/mL) for 1 h with or without

218

treatment of the tested phytochemicals (10 µM) for 6 h. Cells were grown with primary rabbit

219

monoclonal NF-κB p65 antibody and anti-rabbit alexa 488 overnight at 4 ˚C. Nuclei were

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counterstained with 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride, and cells were 10 ACS Paragon Plus Environment

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visualized using confocal microscopy at a 63× magnification (TCS-Sp5, Leica microsystem,

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Wetzlar, Germany).

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Cecal Ligation and Puncture (CLP). To simulate severe sepsis, male mice were

224

anesthetized as described in the previous section In vivo permeability and leukocyte

225

migration assay. They were allowed to breathe spontaneously during operations. CLP-

226

induced sepsis was implemented with reference to a previous procedure.22 In brief, a 2-cm

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midline incision was made to expose the cecum and adjoining intestine. The cecum was

228

firmly ligated with a 3.0-silk suture at 5.0 mm from the cecal tip and punctured once with a

229

22-gauge needle. Gentle squeezing was then performed to extrude a small amount of feces

230

from the perforation site, and returned to the peritoneal cavity. The laparotomy site was

231

carefully sutured using 4.0-silk. In sham control groups, the cecum was exposed but not

232

ligated or punctured and returned to the abdominal cavity. This protocol was approved by the

233

Animal Care Committee at Kyungpook National University.

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Statistical Analysis. Results are conveyed as mean ± standard deviation (SD) of at

235

least three independent experiments. Statistical significance was established using analysis of

236

variance (ANOVA; SPSS, version 14.0, SPSS Science, Chicago, IL, USA) and p-values less

237

than 0.05 (*p < 0.05) were considered significant. Statistical significance between two groups

238

was determined by Student’s t-test and survival analysis of CLP-induced sepsis outcomes

239

was conducted using Kaplan–Meier analysis.

240 241

RESULTS AND DISCUSSION

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Structural Elucidation of the New Secondary Metabolites. From the EtOAc

243

extract exhibiting potent inhibitory activity of HMGB1 release,13 compounds 1–6 were

244

isolated and identified using various chromatographic, spectroscopic, and spectrometric

245

techniques (Figure 1). Compound 1 was obtained as a yellow amorphous powder. The

246

molecular formula was established as C16H14O5 based on the HRESIMS sodium adduct ion at 11 ACS Paragon Plus Environment


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m/z 309.1623 (calcd [M+Na]+, m/z 309.1651) and 13C NMR data (Table 1). The presence of

248

hydroxy, carbonyl, and aromatic moieties was deduced from IR absorption at 3306 and 1656,

249

and 1504, 1471, and 1458 cm-1, respectively. The UV spectrum displaying absorption bands

250

at 237, 279, and 330 (sh) nm implies that compound 1 possesses a flavanone core structure.17

251

The 1H and

252

diastereotopic methylene group (δH 2.82, 2.90; δC 43.9), a methine group (δH 5.73; δC, 76.7),

253

a ketocarbonyl group (δC 193.8), and 1,2,4,5-tetrasubstituted (δH 7.31, 6.48; δC 104.7, 108.1,

254

113.7, 145.2, 156.9, 160.6) and 1,2-disubstituted aromatic moieties (δH 6.83, 6.91, 7.17, 7.49;

255

δC 116.2, 120.7, 127.2, 127.6, 130.2, 155.2), which also evidences the presence of the

256

flavanone architecture of 1. The HMBC correlation between the methine proton (δH 5.73) and

257

an oxygenated aromatic carbon (δC 155.2) along with COSY correlations of the aromatic

258

protons (δH 6.83, 6.91, 7.17, 7.49) confirmed the presence of the flavanone moiety where C-2

259

of the B-ring was substituted with a hydroxy group (Figure 2A). The substitution of the A-

260

ring was established based on the coupling pattern of the two aromatic protons at δH 6.48 and

261

7.31 as singlets and HMBC correlations from the methoxy protons (δH 3.87) to an oxygenated

262

tertiary carbon (δC 156.9) and from one of the aromatic protons (δH 7.31) to the ketocarbonyl

263

carbon (δC 193.8). The absolute configuration of 1 was established utilizing electronic

264

circular dichroism (ECD) analysis. Based on the ECD spectrum the negative Cotton effect for

265

the π → π* transition at ca. 250 nm (Figure 2B) was observed, which corroborated that 1

266

possesses the 2S configuration for 1.18 Consequently, compound 1 was identified as 2S-2',7-

267

dihydroxy-6-methoxyflavanone.

268

13

C NMR data of 1 (Table 1) exhibited resonances and coupling patterns for a

Compound 2 was obtained as a yellow amorphous powder. The HRESIMS spectrum of 2

269

exhibiting a sodium adduct ion at m/z 323.0895 (calcd [M+Na]+, m/z 323.0895) and its

13

270

NMR data led the molecular formula to be assigned as C17H16O5. The pronounced IR (3414,

271

1651, 1502, 1455, 1438 cm-1) and UV (237, 275, 337 nm (sh)) bands of 2 are similar to those

272

of 1, which suggests a structural resemblance between the two compounds. The 1H and 13C

C


Page 13 of 35


273

NMR data of 2 (Table 1) were analogous to those of 1 except for the presence of an

274

additional O-methyl group deduced from its characteristic chemical shift values (δH 3.81, δC

275

56.8). This indicates that compound 2 is structurally based on a flavanone backbone with

276

substitution of one of the rings being slightly different from 1. The respective HMBC

277

correlations from two sets of methoxy protons (δH 3.81, 3.88) to oxygenated tertiary aromatic

278

carbons (δC 146.1, 158.4) indicated that C-6 and C-7 of the A-ring in 2 were substituted with

279

methoxy groups (Figure 2A). The ring assignment was confirmed by those methoxy protons

280

(δH 3.81, 3.88) showing NOESY correlations with the aromatic protons (δH 7.27, 6.46, Figure

281

S15). The absolute configuration of C-2 was determined to be S based on the ECD data

282

similar to those of 1 (Figure 2B).18 Therefore, the structure of compound 2 was defined as 2S-

283

2'-hydroxy-6,7-dimethoxy-flavanone.

284 285

Compound 3 was identified as 2S-5,2'-dihydroxy-6,7-methylenedioxyflavanone by comparison of its observed and reported NMR and MS data.19

286

Compound 4 was obtained as white crystals. The IR spectrum exhibited absorption bands

287

at 1113, 1332, 1428, 1471, 1635, and 3089 cm-1, which is indicative of the presence of

288

hydroxy, carbonyl, aromatic, and ethereal functionalities. The molecular of 4 was established

289

as C11H10O5 based on an HRESIMS spectrum displaying a sodium adduct ion at m/z

290

245.0426 (calcd [M+Na]+, m/z 245.0430) and 13C NMR data of 4. The 1D NMR data (Table

291

1) were similar to those of the known chromone (6)20 except for

292

oxygenated tertiary aromatic carbon at δC 135.0. Two methoxy group protons (δH 3.92, 3.98)

293

exhibiting HMBC correlations with respective oxygenated tertiary aromatic carbons (δC

294

145.92, 135.0) and an aromatic proton (δH 7.30) displaying such a heteronuclear correlation

295

with an allylic carbonyl carbon (δC 176.8) led to the characterization of compound 4 (Figure

296

2A) as 7-hydroxy-6,8-dimethoxychromone. Even though this compound has been

297

commercially available, it has not been isolated from natural sources, which leads the

298

compound to be reported as a new natural product in the current study.

the presence of an



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299

Compounds 5 and 6 were characterized as 6-methoxychromanone and 6,7-

300

dimethoxychromone, respectively based on the comparison of their observed NMR data with

301

those reported in the literature 20,21.

302

Inhibitory Potential of Purified Compounds on LPS and CLP-Induced

303

HMGB1 release. Endothelial cells (ECs) play a central role in sustaining barrier integrity as

304

a selective barrier between the blood and underlying tissue interstitium and the release of

305

HMGB1 is activated in ECs with the presence of lipopolysaccharide (LPS) stress.22,23 The

306

HMGB1 release induces the disruption of vascular barrier integrity, which consequently

307

accelerates leucocytes to access inflamed tissue and prompts vascular hyperpermeability.24

308

Such vascular disorder ultimately causes vascular inflammatory manifestations including

309

septic complications.4,24 LPS-mediated HMGB1 release occurred with late kinetics of eight

310

hours after LPS insult and reached its maximal level after 16 hours.25 Based on these studies

311

the flavanones and chromones from S. herbacea (1–6) were evaluated for their inhibitory

312

potential of LPS-stimulated HMGB1 release. Human umbilical vein endothelial cells

313

(HUVECs) were stressed with LPS (100 ng/mL) for 16 hours and treated with various

314

concentrations of those secondary metabolites for six hours. The release of HMGB1 upon the

315

LPS insult was monitored employing the enzyme-linked immunosorbent assay (ELISA). The

316

LPS stress (100 ng/mL) elevated the release of HMGB1 (Figures 3A and 3B), which is in

317

accordance with previous studies.25,26 The tested compounds alone did not stimulate HMGB1

318

release. Treatment with 5 µM of compounds 1–6 caused inhibition of LPS-stimulated

319

HMGB1 secretion and 10 µM administration reduced the LPS-induced release by half

320

(Figures 3A and 3B). To confirm the in vitro inhibitory activity on HMGB1 release in vivo,

321

those compounds were tested using cecal ligation and puncture (CLP)-induced septic murine

322

models.27 Assuming that the average weight of a mouse is 20 g and an average blood volume

323

is 2 mL, the injected amount of compounds is equivalent to 1, 2, 5, or 10 µM in peripheral

324

blood. The results shown in Figures 3C and 3D revealed that the addition of the purified 14 ACS Paragon Plus Environment

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325

compounds reduced HMGB1 release by 1.5- and 2.0-fold at 5 µM and 10 µM, respectively.

326

Among these active compounds, treatment of murine models with compound 4 exhibited the

327

most potent inhibitory activity against CLP-induced HMGB1 secretion in vivo (Figure D).

328

Previous studies verified that released HMGB1 interacts with at least three pathogen-

329

associated cell surface pattern recognition receptors including toll-like receptors (TLR) 2 and

330

4, and the receptor for advanced glycation endproducts (RAGE).28,29 This interaction

331

consequently triggers tumor necrosis factor-(TNF)-α and nuclear factor-(NF)-κB activation in

332

target cells.28 With reference to these studies, HUVECs were stimulated by LPS to release

333

HMGB1 and enhance the expression of HMGB1-associated receptors. Compounds 1–6 were

334

then added to the stressed HUVES and assessed for their in vitro suppressive activity on the

335

receptor expression using ELISA. While LPS-induced HMGB1 release up-regulated

336

expression of those cell surface pattern recognition receptors 5.0-fold, each compound (10

337

µM) mitigated the enhanced expression 2.5-fold (Figure S23). This result infers suppressive

338

potential of the tested compounds against HMGB1 vascular stress via down-regulating the

339

expression of associated receptors. These anti-HMGB1 compounds were validated to be non-

340

cytotoxic up to 20 µM in HUVECs (Figures S24).

341

Restoring Effect on HMGB1-Stimulated Vascular Barrier Disruption. After

342

compounds 1–6 were validated to show anti-release activity of HMGB1, further mechanistic

343

studies were carried out to investigate whether those active compounds can protect vascular

344

barrier disruption created by HMGB1 release. The LPS and HMGB1 insults increased

345

vascular permeability by 4.0- and 5.0-fold, respectively, as compared with the DMSO-treated

346

negative control (Figure 4A). The treatment of HUVECs with 10 µM of each compound for

347

six hours after addition of LPS (100 ng/mL) or HMGB1 (1 µg/mL) reduced the vascular

348

hyperpermeability (Figure 4A) to the levels of the negative control, which demonstrated that

349

the tested phenolic compounds restored vascular integrity. To confirm the observed in vitro

350

vascular protecting action of those compounds utilizing in vivo models, disruption of 15 ACS Paragon Plus Environment


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351

endothelial permeability in murine models was conducted upon HMGB1 or CLP stimuli and

352

dye leakage into the peritoneum was assessed based upon the amounts of Evans blue in

353

peritoneal washings. In accordance with the in vitro protective effects of compounds 1–6

354

against vascular disruption, treatment with each compound (10 µM) alleviated the dye

355

leakage, initiated by HMGB1- or CLP-stimulated barrier integrity dysfunction, into the

356

peritoneum (Figure 4B). Based on these data, the secondary metabolites from S. herbacea

357

exerted protective action of vascular barrier integrity against endothelial vascular disruption

358

caused by pro-inflammatory vascular insults.

359

Suppressive Effects on HMGB1-Triggered CAMs Expression, THP-1

360

Adhesion, and Migration. As briefly addressed in the previous section, HMGB1

361

coordinates with the pathogenic cell surface pattern recognition receptors to facilitate pro-

362

inflammatory responses, which stimulates cell surface expression of cell adhesion molecules

363

(CAMs) including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion

364

molecule-1 (VCAM-1), and E-selectin. These activated CAMs mediate adhesion and

365

migration of leukocytes across the endothelium to inflamed sites.5,30 Thus, VCAM-1, ICAM-

366

1, and E-selectin levels were up-regulated by HMGB1 and the levels were evaluated upon

367

treatment of HUVECs with the secondary metabolites (1–6) to investigate whether those

368

compounds exert suppressive activity of pro-inflammatory leukocyte migration via inhibiting

369

the enhanced CAM expression. HMGB1 increased the expression of VCAM-1, ICAM-1, and

370

E-selectin in HUVECs 4.5-fold as compared to the negative control with treatment of DMSO.

371

Treatment of the test cells with 10 µM of each compound diminished the heightened

372

expression 2.3-fold as shown in Figure 5A. A cell-cell adhesion assay employing the

373

monocytic cell line THP-1 was successively implemented to probe whether the purified

374

compounds mitigate the strengthened CAMs expression. As Figure 5B depicts, the addition

375

of those phytochemicals reduced THP-1 cell adherence towards HMGB1-treated HUVECs

376

2.3-fold as compared to the adherence level detected at HMGB1-challenged HUVECs. 16 ACS Paragon Plus Environment

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377

CAMs also influence migration of leukocytes to the sites of inflammation,30 which leads the

378

evaluation of the compounds for their suppressing potential against leukocyte migration. In

379

agreement with the adhesion assay, the test compounds at 10 µM hampered HMGB1-induced

380

migration of leukocytes by 2.2-fold (Figure 5C). These data verified that the purified

381

compounds inhibit not only CAMs expression but also the adherence of leukocytes to the

382

HUVECs, which finally impedes subsequent migration (Figures 5B and 5C). The in vitro

383

inhibitory potential of the examined compounds on leukocytes adherence and migration was

384

verified in vivo using murine models. Animals were stimulated by HMGB1 or CLP and

385

administered with compounds 1–6, and migrated leukocytes into their peritoneal cavities

386

were counted. The migration of leukocytes escalated upon the HMGB1 and CLP vascular

387

stimuli 7.0- and 8.0-fold as compared to the DMSO-treated group. Intravenous administration

388

of mice with the active phytochemicals (10 µM ) attenuated the transendothelial migration

389

stimulated by HMGB1 by 1.7- to 2.3-fold and induced by CLP by half as shown in Figure 5D.

390

These studies inferred that the active compounds inhibit the adhesion and migration of

391

leukocytes to inflamed endothelium via hindering expression of adherence factors induced by

392

pro-inflammatory conditions.

393

Suppressive Activity on HMGB1-Stimulated Generation of Cytokines and

394

MAPKs. Phosphorylation and translocation of NF-κB are prerequisites for HMGB1-

395

triggered inflammatory responses in terms of the fact that NF-κB activation is involved in

396

promoting the expression of CAMs and up-regulating pro-inflammatory cytokines such as

397

TNF-ɑ and interleukin 1β (IL-1β).31-33 The mitogen-activated protein kinase (MAPK)

398

extracellular regulated kinase (ERK) 1/2 and p38 are also stimulated in inducing vascular

399

inflammatory responses against HMGB1 stress.34,35 These studies led us to examine the

400

inhibitory potential of the purified compounds on production of those pro-inflammatory

401

factors. HUVECs were stressed with HMGB1 and treated with each compound for six hours

402

and expression of the pro-inflammatory factors was evaluated employing ELISA. The levels 17 ACS Paragon Plus Environment


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403

of TNF-α and IL-1β increased upon HMGB1 stress 3.3-fold and 5.0-fold, respectively and

404

the treatment with 10 µM of each compound resulted in decreasing the enhanced levels 2.0-

405

and 1.6-fold, respectively (Figures 6A and 6B). The phosphorylation of ERK1/2 and p38 also

406

showed similar patterns in the expression upon HMGB1 stress and subsequent addition of the

407

active molecules (Figures 6C and 6D).

408

For the assessment of suppressive activity of these active molecules on NF-κB, which is

409

upstream of the TNF-α and IL-1β pathways, the translocation of NF-κB from cytosol to

410

nucleus was evaluated by assessing the expression of the NF-κB p65 protein (p65), the active

411

subunit of the NF-κB complex. According to the results of the ELISA data shown in Figure

412

7A, the phosphorylation of p65 expression was elevated 5.0-fold as compared with the

413

negative control and the level was decreased by half upon treatment with all the tested

414

compounds. This implies that the tested compound evidently inhibited the translocation of

415

NF-κB to the nucleus. The total p65 expression did not exhibit a pronounced change with or

416

without HMGB1 insult and administration with the active compounds (Figure 7A). To

417

validate the observed anti-translational activity, p65 nuclear fluorescence was measured

418

(Figure 7B). The fluorescence intensity increased upon HMGB1 insult and the treatment of

419

the purified phytochemicals (10 µM) alleviated the NF-κB nuclear translocation by a similar

420

extent based on Figure 7B. Immunofluorescence (IF) microscopy analysis with compound 5,

421

exhibiting slightly more powerful anti-translational activity (Figure 7B), was implemented

422

using the p65 and fluorescein isothiocyanate (FITC)-conjugated antibody to visualize the

423

translocation of NF-κB p65 (Figure 7C). The appreciation of HMGB1-stimulated pro-

424

inflammatory stress rendered an increase in translocation of p65 into the nucleus based on the

425

overlaid image of p65 (red) and 4′,6-diamidino-2-phenylindole (DAPI, blue) commonly used

426

for staining of nuclei (Figure 7C, second lane).36 The pre-incubation with compound 5 (10

427

µM) indeed mitigated the translocation of p65 on the basis of the decreased fluorescence

428

intensity in the nuclei and enhanced intensity in the cytoplasm, which can be deduced from 18 ACS Paragon Plus Environment

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the nuclear staining by DAPI (Figure 7C, third lane). The IF staining assay outcome is

430

accordance with the aforementioned ELISA (Figure 7A) and nuclear fluorescence (Figure 7B)

431

results. These assays collectively confirmed the inhibitory potential of the tested bioactives

432

on the NF-κB translocation.

433

These data supported that the active compounds alleviated HMGB1-induced endothelial pro-

434

inflammatory stress via curbing pro-inflammatory pathways involved in the transcriptional

435

factors, cytokines, and MAPK cascades.

436

Improvement of Survival Rate Against Septic Mortality. To evaluate whether the

437

active phenolic compounds from S. herbacea can improve severe septic mortality, they were

438

administered to CLP-operated mice, an in vivo model simulating severe septic conditions.27

439

Initially, administration of the CLP models with compounds 1–6 (5.7–6.0 µg/mouse, 12 h

440

after CLP) did not prevent septic death (data not shown), which leads to a change of the times

441

of administration to the septic models, i.e., two times, at 12 and 50 hours after CLP. This

442

alternative dosing strategy improved the survival rate from 40 to 60% according to a Kaplan-

443

Meier survival analysis (Figures 8A and 8B) as such a dosing strategy has been proven to be

444

more effective in our previous studies as well.13,15 Importantly, compound 4, structurally

445

based on the chromone scaffold, displayed the most effective activity in preventing the septic

446

lethality. The pharmacophore has not been investigated for potential application for the

447

alleviation of septic fatalities. This, together with the examined flavanone showing the

448

observed improvement of survival rate, may warrant further efforts for such bi- and tricyclic

449

architecture to be developed into drug leads and food additives or supplements for

450

prevention/ treatment of severe septic manifestations or complications.

451

Even though flavonoids and chromones are abundant plant-derived secondary

452

metabolites, they have been rarely investigated for their potential to avert pathogenic

453

conditions related to endothelial hyperpermeability.37,38 This study, identifying flavonoids

454

and chromones from S. herbacea and investigating their inhibitory mechanisms of vascular 19 ACS Paragon Plus Environment


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455

pro-inflammatory factors against vascular barrier disruption, may motivate further isolation

456

and/ or semi-synthesis using such enriched and nontoxic natural product scaffolds for food

457

formulations or additives targeting pathogenic conditions associated with severe vascular

458

inflammatory diseases.

459

ASSOCIATED CONTENT

460

Supporting Information

461 462 463 464 465 466 467

NMR spectral data, IR and HRESIMS spectra. The Supporting Information is available free of charge on the ACS Publications website at DOI: AUTHOR INFORMATION Corresponding Authors * Tel: +82-42-821-5925. Fax: +82-42-823-6566. E-mail: [email protected] (M. Na). * Tel.: +82 53 950 8570. Fax: +82 53 950 8557. E-mail: [email protected] (J.-S. Bae).

468

Author Contributions

469

#

470 471

Notes The authors declare no competing financial interest.

472

ACKNOWLEDGMENTS

473 474 475 476 477 478 479

We recognize Dr. D. Ferreira (University of Mississippi) for his meticulous proof reading. This study was supported by the Basic Science Program (2014R1A2A1A11049526 and 2014R1A2A2A01006793) and Priority Research Centers Program (NRF-2009-0093815) through the National Research Foundation of Korea (NRF) grant funded by a Korean government, and by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI15C0001).

N. Q. Tuan and W. Lee contributed equally.


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1-9.

584

(38) Chang, Y.-C.; Tsai, M.-H.; Sheu, W. H.-H.; Hsieh, S.-C.; Chiang, A.-N., The

585

therapeutic potential and mechanisms of action of quercetin in relation to lipopolysaccharide-

586

induced sepsis in vitro and in vivo. PLoS ONE 2013, 8, e80744.

587 588 589


Page 25 of 35

590 591 592


Table 1. 1H (600 MHz) and 13C NMR (150 MHz) Data of Compounds 1, 2 (methanol-d4) and 4 (CDCl3) 1

position

2

δH, mult (J in Hz) 2 3

5.73, dd (13.0, 3.3) 2.82, dd (17.0, 3.3) 2.90, dd (17.0, 13.0)

δC

4

δH, mult (J in Hz)

δC

δH, mult (J in Hz)

δC

76.7

CH

5.74 d (2.5)

76.9

CH

7.79 d (5.9)

154.5

CH

43.9

CH2

2.80 d (2.3)

43.4

CH2

6.23 d (5.9)

112.3

CH

4

-

193.8

-

-

193.8

-

-

176.8

-

5

7.31, s

104.7

CH

6.64 s

101.6

CH

7.30 s

99.5

CH

6

-

156.9

-

-

158.4

-

-

145.9

-

7

-

145.2

-

-

146.1

-

-

144.1

-

8

6.48, s

108.1

CH

7.27 s

107.9

CH

-

135.0

-

9

-

160.6

-

-

160.6

-

-

146.6

-

10

-

113.7

-

-

114.1

-

-

117.8

-

1'

-

127.2

-

-

127.0

-

-

-

-

2'

-

155.2

-

-

155.3

-

-

-

-

3'

6.83, dd (8.1, 0.7)

116.2

CH

6.81 dd (8.1, 0.7)

116.2

CH

-

-

-

4'

7.17, m

130.2

CH

7.15 td (7. 9, 1.6)

130.2

CH

-

-

-

5'

6.91, m

120.7

CH

6.87 dd (7.6, 0.7)

120.6

CH

-

-

-

6'

7.49, d (7.7)

127.6

CH

7.49 dd (1.4, 7.7)

127.6

CH

-

-

-

6-OCH3

3.87, s

56.6

CH3

3.88 s

56.6

CH3

3.92 s

56.6

CH3

7-OCH3

-

-

-

3.81 s

56.8

CH3

-

-

8-OCH3

-

-

-

-

-

-

3.98 s

61.7

CH3

25

ACS Paragon Plus Environment


Page 26 of 35

R3 HO RO

7

8

O

9

A H3CO

6

HO 2' 1'

4

9

O 2

6

6'

R1

O

3 OH

5

7

O

O

2

C 10

B

8

R2

4'

O

3

10 4

5

O

O

1R=H 2 R = CH3

593 594

3

4 R1 = R3 = OCH3, R2 = OH 5 R1 = OCH3, R2 = R3 = H 6 R1 = R2 = OCH3, R3 = H

Figure 1. Structures of compounds 1–6 from S. herbacea

595


Page 27 of 35


596 HO HO

HO

O

7

H3CO

O

OCH3 HO

9

10

H3CO

4

5

H3CO

H3CO

O

597

1

O

7

4

5

O

O

1

2

598

Figure 2A. Selected HMBC (→) and COSY (

599

clear presentation of correlations.

4

) correlations. Stereodescriptors were omitted for a

600



Page 28 of 35

601

4 4

2 0 -2

2 180

230

280

330

380

-2

-4 -6

602

0

180

230

280

330

380

-4

1

-6

2

Figure 2B. ECD spectrum of compounds 1 and 2

603


Page 29 of 35


250

300

A 1 2 3

200 150

* *

100 50

HMGB1 (ng/mL)

HMGB1 (ng/mL)

300

0 10 LPS

Comp,[µ µM]

2

5

4 5 6

200 150

*

*

100 50

10

D

LPS + Comp, [µ µM] 200

C

150

1 2 3

100

* *

50

10 LPS

Comp,[µ µM]

HMGB1 (ng/mL)

HMGB1 (ng/mL)

1

0

1

2

5

10

LPS + Comp, [µ µM]

D

150

*

4 5 6

100

#

*

50 0

D

10 CLP

Comp,[µ µM]

604 605 606 607 608 609 610 611

B

0 D

200

250

1

2

5

10

CLP + Comp, [µ µM]

D

10 CLP

Comp,[µ µM]

1

2

5

10

CLP + Comp, [µ µM]

Figure 3. Assessment of inhibitory effects on HMGB1 release in vitro (A, B) and in vivo (C, D). (A) HUVECs were treated with the indicated concentrations of 1 (white bar), 2 (gray bar), or 3 (black bar) for 6 h after stimulation with LPS 100 ng/mL for 16 h; HMGB1 release was measured by ELISA. D=0.2% DMSO. (B) As (A) except 4 (white bar), 5 (gray bar) or 6 (black bar). (C) Male C57BL/6 mice underwent CLP and were administered 1 (white bar), 2 (gray bar) or 3 (black bar) intravenously 12 h after CLP (n=5). Serum HMGB1 levels were measured by ELISA. (D) As (C) except 4 (white bar), 5 (gray bar) or 6 (black bar). D=0.2% DMSO. Sham-operated animal groups were utilized for the DMSO- and compounds alone-treated groups. The administered amounts of the compounds at 1, 2, 5, and 10 µM in vivo were as follows; 1 0.57, 1.15, 2.86, and 5.73 µg/mouse; 2 and 3 0.60, 1.20, 3.00, and 6.00 µg/mouse; 4 0.44, 0.89, 2.22, and 4.44 µg/mouse; 5 0.36, 0.71, 1.78, and 3.56 µg/mouse; 6 0.41, 0.82, 2.06, and 4.12 µg/mouse; Results are expressed as the mean ± SD of three independent experiments. *p < 0.05 vs. LPS alone (A, B) or CLP alone (C, D) #p < 0.05 vs. compounds 4 or 5.

29



Permeability (OD650)

0.6

Page 30 of 35

612

A

613 LPS HMGB1 614

0.4

*

0.2

615

*

*

*

*

* 616 617 618

0 D

L/H

1 2 3 4 5 6 619 LPS or HMGB1 + Comp, [10 µM]620 621

Dye Leakage (µ µg/mouse)

6

B

HMGB1 622 CLP 623

4

*

*

*

*

2

*

* 624 625 626

0

627

D 631 632 633 634 635 636 637 638 639 640 641

H/C

1 2 3 4 5 6 628 HMGB1 or CLP + Comp, [10 µM]629 630

Figure 4. Restoring effects of the tested compounds on HMGB1-mediated vascular hyperpermeability in vitro (A, top) and in vivo (B, bottom). (A) HUVECs were stimulated with LPS (white bar, 100 ng/mL for 4 h) or HMGB1 (black bar, 1 µg/mL, 16 h) and treated with each compounds (10 µM ) for 6 h. Permeability was monitored by measuring the flux of Evans blue bound albumin across HUVECs. L=LPS; H=HMGB1. (B) The in vivo effects of intravenously injected each compounds (10 µM/mouse) on HMGB1- (white bar, 2 µg/mouse, intravenous) or CLP- (black bar, at 24 h after CLP) induced vascular hyperpermeability in mice. The restoring activity was examined by measuring the amount of Evans blue in peritoneal washings (µg/mouse, n=5). H=HMGB1; C=CLP. Results are expressed as the mean ± SD of three independent experiments. *p < 0.05 vs. LPS (A), CLP (B) or HMGB1 (A and B)

642



120

A

VCAM ICAM E-Selectin

0.4

*

*

0.2

*

*

*

*

Migration Index

250

80 60

*

*

*

*

1

2

3

4

40

*

*

20

C

200 150

*

100

*

*

*

*

*

50

D

D

2 3 4 5 6 HMGB1 + Comp, [10 µM]

0

644 645 646 647 648 649

B

0

0 D HMGB1 1

643

100

Adherence (%)

0.6

H

1 2 3 4 5 6 HMGB1 + Comp, [10 µM]

H

5

6

HMGB1 + Comp, [10 µM]

Total Leukocytes (x 106)

CAM Expression (OD490)

Page 31 of 35

10

HMGB1 CLP

8 6

*

4

*

*

*

*

*

2 0 D H/C 1 2 3 4 5 6 HMGB1 or CLP + Comp, [10 µM]

Figure 5. Anti-migration activity on HMGB1-mediated pro-inflammatory responses. HUVECs were stimulated with HMGB1 (1 µg/mL) for 16 h followed by treatment with each the compounds (10 µM) for 6 h. HMGB1-facilitated (A) expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) in HUVECs, (B) adherence of monocytes to HUVEC monolayers, and (C) migration of monocytes through HUVEC monolayers were analyzed. (D) Male C57BL/6 mice were stimulated with HMGB1- (white bar, 2 µg/mouse, intravenous) or CLP (black bar, at 24 h after CLP) and were treated with each compound (10 µM). HMGB1- or CLP-mediated migration of leukocytes into the peritoneal cavity of mice was analyzed. H=HMGB1; C=CLP. Results are expressed as the mean ± SD of three independent experiments. *p < 0.05 vs. HMGB1 (A-D) or CLP (D).

31


600 500

Active

A

400

*

*

300

*

IL-1β β Production (pg/mL)

TNF-α α Production (pg/mL)


Total

*

*

*

200 100 0 D

H

1

2

3

4

5

300

Active

B

200

*

Active

Total

4

*

*

*

*

*

*

2

*

3

4

*

*

D

H

1

2

5

6

HMGB1 + Comp, [10 µM]

6

Active

D

4

*

*

*

*

Total

*

*

2

0

0 D

H

1 2 3 4 5 6 HMGB1 + Comp, [10 µM]

D HMGB1 1

650 651 652 653

*

0

6

p-p38 Expression (fold increase)

ERK1/2 (Fold Increase)

C

*

Total

100

HMGB1 + Comp, [10 µM] 6

Page 32 of 35

2 3 4 5 6 HMGB1 + Comp, [10 µM]

Figure 6. Suppressive action of 1–6 on HMGB1-stimulated pro-inflammatory factors. HUVECs were stimulated with HMGB1 (1 µg/mL) for 16 h followed by treatment with each compounds (10 µM) for 6 h. HMGB1 (1 µg/mL)-mediated production of TNF-α (A), IL-1β (B), and phosphor (active, white bar) or total (black bar) ERK1/2 (C) and p38 (D) in HUVECs was assessed using ELISA. Results are expressed as the mean ± SD of three independent experiments.

32


Page 33 of 35

654 655 656 657


Figure 7 (A) HUVECs were activated with HMGB1 (1 µg/mL) for 1 h and treated with each compounds (10 µM) for 6 h. The resultant production of phosphor – (white bar) or total p65 (black bar) in HUVECs was evaluated. (B) Quantification of p65 nuclear translocation and (C) IF microscopy analysis of the nuclear translocation of p65 in HUVECs. HUVECs were stimulated for 1 h with 1 µg/mL HMGB1 and treated with 10 µM of all the compounds for (B) and compound 5 for (C) for 6 h. For (C), DAPI was used for nuclear staining.

33



Page 34 of 35

A

Survival

1

Sham

0.8 0.6 0.4

Comp 1* Comp 2* Comp 3*

0.2 0 0

20

CLP

40 60 80 100 [Time after CLP], Hours

120

140

B

Survival

1

Sham

0.8 0.6 0.4

Comp 4* Comp 5* Comp 6*

0.2 0 0 658 659 660 661 662 663 664 665

20

CLP

40 60 80 100 [Time after CLP], Hours

120

140

Figure 8. Improvement of septic lethality upon administration of phenolic compounds. Male C57BL/6 mice (n = 20) were administered intravenously. Each phenolic derivative (10 µM) was injected intravenously at 12 h and 50 h after CLP. Animal survival was monitored every 6 h after CLP for 126 h. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 10). A Kaplan–Meier survival analysis was used for determination of overall survival rates vs. CLPtreated mice. *p < 0.00001 vs. CLP.


Page 35 of 35

666


TABLE OF CONTENTS GRAPHICS

667 668


Flavanones and Chromones from Salicornia herbacea Mitigate Septic

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