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Polyphenols from blossoms of Citrus aurantium L. var. amara Engl. show significant anti-complement and anti-inflammatory effects Chun-Yan Shen, Jian-Guo Jiang, Chun-Ling Huang, Wei Zhu, and Chao-Yang Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03759 • Publication Date (Web): 24 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Journal of Agricultural and Food Chemistry

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Polyphenols from blossoms of Citrus aurantium L. var. amara Engl.

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show significant anti-complement and anti-inflammatory effects

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Chun-Yan Shen †, Jian-Guo Jiang *†, Chun-Ling Huang § ⊗, Wei Zhu § , Chao-Yang Zheng *§

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†

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510640, China

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§

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510120, China

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⊗

College of Food and Bioengineering, South China University of Technology, Guangzhou,

The second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou

Sci-tech Industrial Park, Guangzhou University of Chinese Medicine, Guangzhou 510120, China

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*Author (Jian-Guo Jiang) for correspondence (e-mail: [email protected]; phone +86-20-

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87113849; fax: +86-20-87113843)

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*Author (Chao-Yang Zheng) for correspondence ([email protected]; phone +86-20-

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39318571; fax: +86-20-39318571)

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ACS Paragon Plus Environment


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Abstract

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Citrus aurantium L. var. amara Engl. (CAVA) was traditionally used as a digestant or expectorant

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in China. Crude polyphenols (CAVAP-W) extracted from blossoms of CAVA were mainly

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composed of eriocitrin/neoeriocitrin, eriocitrin/neoeriocitrin, rhoifolin, hesperidin, naringin,

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rutin, veronicastroside, neohesperidin and hesperetin by LC-MS analysis. CAVAP-W showed

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significant anti-complement and anti-inflammatory effects. Due to the close relationship between

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anti-complement and anti-inflammatory activity, the anti-inflammatory effect was further

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investigated and the results showed that CAVAP-W significantly suppressed production of

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interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and mRNA

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expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, IL-1β and cyclooxygenase-2

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(COX-2) in lipopolysaccharides-stimulated RAW264.7 cells. Furthermore, CAVAP-W inhibited

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mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation through

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suppressing nuclear translocation of nuclear factor-kappa B (NF-κB) P65, degradation and

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phosphorylation of IκBα, phosphorylation of IκKBɑ/ß, c-Jun N-terminal kinase (JNK) and P38,

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and activation of COX-2, thereby exerting the anti-inflammatory effects.

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Key words: Citrus aurantium L. var. amara Engl.; polyphenolys; LC-MS; anti-complement; anti-

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inflammatory

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INTRODUCTION

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Complement system, playing a critical role in the host defense, had various beneficial

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biological activities, such as protecting against infectious agents and opsonizing anti-genic

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particles. However, excessive activation of complement could also induce severe tissue damage

40

and various inflammation associated diseases. Inflammation was a complex process regulated by

41

many pro-inflammatory mediators and their excessive production might lead to various acute and

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chronic inflammatory diseases including atherosclerosis, rheumatoid arthritis and even cancer.1-3

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There is growing acceptance that active ingredients extracted from natural products usually

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exert various beneficial effects such as anti-aging, immunomodulatory, anti-inflammatory and

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anti-cancer, with few side effects.4-7 Specifically, polyphenols are a major class of phytochemicals

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found in natural products and receiving much attention owing to their great contributions to

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human health. Especially, a growing number of published data focused on the anti-inflammatory

48

effects of Citrus polyphenols.8-11 For example, Kang et al. found that the flavonoids extracted

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from Korea Citrus aurantium L. displayed significant anti-inflammatory effects on LPS-induced

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RAW264.7 cells by suppressing mitogen-activated protein kinase (MAPK) and nuclear factor-

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kappa B (NF-κB) signaling pathways.10

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Citrus aurantium L. var. amara Engl. (CAVA), a member of genus Citrus (Rutaceae), was

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widely used as edible and medicinal resource nowadays in China. It was traditionally used as a

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digestant and expectorant in China. Also, blossoms of CAVA were claimed to be a popular kind

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of beverage. Our published reports

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oil, flavonoids and polysaccharides extracted from blossoms of CAVA possessed various

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pharmacological activities.

12-14

showed that many active ingredients including essential

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Nevertheless, information about possible bioactivities of its polyphenols was rather limited.

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Therefore, this study was undertaken to investigate the anti-complement and anti-inflammatory

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activities of polyphenols from CAVA as well as explore the underlying molecular mechanism.

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Simultaneously, high performance liquid chromatography-mass spectrometry (LC-MS) analysis of

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CAVAP-W was also performed in order to speculate the specific constituents responsible for the

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observed bioactivity.

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MATERIALS AND METHODS

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Materals and Chemicals

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Blossoms of Citrus aurantium L. var. amara Engl. were collected in the year of 2016 from

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Zhejiang Province in China. The blossoms were washed, drained, freeze-dried and powdered with

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a grinder and then packed into sealed containers for further use. Dulbecco’s modified eagle

69

medium (DMEM), fetal bovine serum (FBS), Trizol Reagent and Penicillin G/streptomycin were

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purchased from GIBCO (Grand Island, NY). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-

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tetrazolium bromide (MTT) and lipopolysaccharides (LPS) were obtained from Sigma-Aldrich (St.

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Louis, MO, USA). NF-κB activation-nuclear translocation assay kit and the nuclear and

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cytoplasmic protein extraction kit were purchased from Beyotime Biotech (Guangzhou, China).

74

ELISA kits of mouse interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-1β

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(IL-1β) were purchased from Cusabio Biothch CO., Ltd. (Wuhan, China). The antibodies to

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GAPDH, phospho-SAPK/JNK, phospho-P38 MAPK, NF-κB P65, IκBα, phospho-IκKα/ß and

77

cyclooxygenase-2 (COX-2) were all purchased from Cell Signaling Technology (Beverly, MA,

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USA). Acetonitrile was HPLC-grade and all other reagents used in this research were of analytical

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grade.

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Extraction of Polyphenols

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Processed powders (5.0 g) of CAVA blossoms were extracted at 90 oC for 1 h with 100 mL of

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distilled water. The resulting extracts were filtered through filter papers and evaporated under

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reduced pressure using a rotary vacuum evaporator at 40 oC. The dried samples were eventually

84

obtained and named as CAVAP-W.

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Determination of Total Polyphenols Contents

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The dried samples (CAVAP-W) obtained in 2.2 were used for determining total phenolic

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contents according to the published method.15 Aliquots (0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mL) of the

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sample solutions were taken in separated test tubes and made up to the volume of 1 mL with

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distilled water. Then 0.5 mL of Folin–Ciocalteu reagent and 2.5 mL of saturated sodium carbonate

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solution were added sequentially to each tube. After vigorous vortex, the tubes containing the

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reaction mixture were placed in the dark for 40 min. Eventually, the absorbance at 725 nm was

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recorded against the blank. Then the total phenolic contents were calculated as gallic acid

93

equivalents according to the calibration curve.

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LC-MS Analysis

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In order to determine the compounds in CAVAP-W, LC-MS analysis was conducted using Agilent Technology 6130 Quadrupole LC/MS equipment based on previous reports.16-18

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The chromatographic separation of CAVAP-W was carried out by HPLC using a Waters

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AQUITY UPLC CSH C18 column (2.1×100 mm, 1.7 µm). The mobile phases were 0.1% aqueous

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formic acid (eluent A) and acetonitrile (eluent B). The eluent gradient applied for solvent B was as

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follows: 0.01-8 min, 2 %-20% B; 8-13 min, 20%-25% B; 13-15 min, 25%-25% B; 15-22 min,

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25%-50% B; 22-25 min, 50%-95% B; 25-28 min, 95%-2% B; 28-30 min, 2% B, at a flow rate of

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0.3 mL/min. The column temperature was 40 oC and the injection volume was 10 µL. MS

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operation parameters were set as follows: capillary voltage, 4000 V; nebulizer pressure, 40 psi;

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drying gas flow rate, 10 L/min; gas temperature, 500 oC. And the electrospray mass spectra

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ranging from m/z 100 to 1000 were taken.

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Anti-complement Activity Assay

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In the current research, distilled water was employed to resuspend CAVAP-W. anti-

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complement activity of CAVAP-W was investigated using the classical pathway according to Seo

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et al. with slight modifications.19 Normal human serum was used as the complement source and

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heparin sodium was used as the positive control.

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Anti-inflammatory Activity Assay

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Cell Culture and Treatment

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Murine macrophage RAW264.7 cell line, human hepatocyte cells LO2 were purchased from

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cell bank of the Chinese Academy of Sciences in Shanghai of China and cultured in DMEM

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supplemented with 10% FBS at 37 oC in a 5% CO2 humidified atmosphere. CAVAP-W was

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dissolved in dimethyl sulfoxide (DMSO), filtered through 0.25 µm filter membrane, and stored at -

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20 °C for future use. During the experiment, the samples were diluted in DMEM to various

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working concentrations varying from 15.625 to 500 µg/mL.

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Assay of Cell Viability

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Aliquot of 100 µL cells (LO2 and RAW264.7) were seeded into 96-well plates at a

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concentration of 7×104 cells/mL and incubated for 24 h to make the cells adherent. Then the

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supernates were removed and fresh medium containing different concentrations of CAVAP-W

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(31.25, 62.5, 125, 250 and 500 µg/mL) were added. After incubation for 24 h, the supernates were

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discarded and 200 µL of MTT solution (0.5 mg/mL) diluted in fresh DMEM without FBS was

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added. 4 h later, MTT solutions were removed and the adherent cells were washed twice with PBS.

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Then 150 µL of DMSO was carefully added into each well to complete dissolve the purple

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formazan crystals. Finally, the absorbance of 570 nm was detected using a microplate reader.

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Determinaton of NO Production and Cytokines Secretion

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Production of NO was determined based on the accumulation of nitrite in the culture

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supernatants using the protocol described by our previous report.7 Briefly, 1 mL of RAW264.7

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cells at a density of 3×105 cells/mL were seeded into each well of 24-well plates and stimulated

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with culture medium or LPS (1 µg/mL) or LPS in the presence of different concentrations of

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CAVAP-W (15.625, 31.25, 62.5, 125, 250 and 500 µg/mL) for 24 h. Then the culture supernatants

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were collected and incubated with equal volume of Griess reagents for 10 min in the dark.

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Eventually, nitrite concentration was determined based on the absorbance at 550 nm and the

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sodium nitrite standard curve. Furthermore, the cytokines (IL-6, TNF-α and IL-1β) released to the

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culture medium, were also determined by ELISA using commercial reagent kits following the

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manufacturer’s instructions. DXM (dexamethasone) is a steroid that can inhibit the expression of

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several immune mediators and widely used in clinical trials for inflammation-related diseases.20

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Therefore, we choose DXM as a positive control when evaluating the anti-inflammatory potential

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of CAVAP-W.

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RT-PCR Analysis

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RT-PCR analysis was conducted according to Lu et al with slight modification.21 In brief,

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RAW264.7 cells were seeded into 6-well plates at 5×105 cells/well and stimulated with tested

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samples for 3 h. Then LPS (1 µg/mL) was added and the cells were incubated for another 12 h. 12

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h later, RAW264.7 cells were washed with cold PBS twice and the total RNA was isolated using

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Trizol reagent. Thereafter, total RNA was quantified with Nano Drop spectrophotometer (Nano

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Drop Technologies, Wilmington, DE) and reverse-transcribed into cDNA with cDNA synthesis

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kits. The resulting cDNA was further amplified by PCR with Fast Star DNA Master SYBR Green

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I

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TTTGTCAAGCTCATTTCCTGGTATG-3, reverse, 5-TGGGATAGGGCCTCTCTTGC-3),

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inducible nitric oxide synthase (iNOS) (forward, 5-CGGCAA ACATGACTTCAGGC-3, reverse,

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5-GCACATCAAAGCGGCCATAG-3), IL-6 (forward, 5-TACTCGGCAAACCTAGTGCG-3,

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reverse,

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GGGGATTATGGCTCAGGGTC-3,

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(forward,

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TGTCCATTGAGGTGGAGAGCTTTCAGC-3)

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ATCCTTGCTGTTCC-3,

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initiated at 50 oC for 2 min, followed by 95 oC for 10 min, 40 cycles of 95 oC for 15 s, 60 oC for 1

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min, 95 oC for 15 s, 60 oC for 1 min and 95 oC for 15 s. The results were expressed as the ratio of

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optimal density normalized to GAPDH.

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Detection of NF-κB Localization by Immunofluorescence

kit

and

5

the

gene-specific

primers

were

as

below:

–GTGTCCCAACATTCATATTGTCAGT-3), reverse,

TNF-α

(forward,

(forward,

5-CGAGGCTCCAGTGAATTCGG-3),

5-TGAAGGGCTGCTTCCAAACCTTTGACC-3,

reverse,

GAPDH

and

COX-2

reverse, (forward,

5-

5IL-1β 5-

5-CAGCAA

5-TGGGCAAAGAATGCAAACATC-3). Cycling was

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The detection of NF-κB nuclear translocation was performed based on the published research

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using the NF-κB Activation-Nuclear Translocation Assay Kit (Beyotime Biotech, China).22

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Initially, RAW264.7 cells adherent on glass coverslips were stimulated with LPS for 3 h and then

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CAVAP-W (500 µg/mL) for another 12 h. When incubation time was reached, cells were fixed

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for15 min and blocked for 1 h at room temperature. Then the primary rabbit anti-NF-κB P65

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antibody and the secondary Cy3-labeled antibody were added successively. Afterwards,

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RAW264.7 cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride

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(DAPI) at 2 µM for 5 min. Each step above was followed by washing three times with washing

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buffer for 5 min each. Finally, RAW264.7 cells were preserved in anti-fade mounting medium and

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observed with a fluorescence microscope immediately.

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Western Blot Analysis

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RAW264.7 cells treated with CAVAP-W at concentrations of 125, 250 and 500 µg/mL were

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scraped into ice-cold PBS and centrifuged at 2,000 g for 5 min. Then the resulting pellets were

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incubated with lysis buffer on ice for 40 min. Vigorous vortex was conducted every 10 min to

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make complete lysis. Then the lysis was centrifuged at 12,000 g for 20 min at 4 °C and the 7



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supernatants were collected as whole cell lysates. Furthermore, the cytoplasmic and nuclear

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proteins were extracted and isolated using the Nuclear and Cytoplasmic Protein Extraction Kit

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(Beyotime, China) based on the manufacturer’s instructions. The concentrations of the proteins

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obtained above were determined using a BCA kit. 40 µg of proteins were separated using 10-12%

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sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to PVDF

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membranes. Then the blots were blocked with 5% BSA diluted in 1×TBST (0.1% Tween-20 in

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1×TBS) at 4 oC overnight, followed with incubation with various primary antibodies and

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horseradish peroxidase-linked secondary antibody. 1×TBST was used to wash the blots after each

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procedure above. The bands were visualized using ECL reagents and quantified with Image J

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software (NIH, USA).

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Statistical Analysis

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All tests were performed in triplicate and the results were presented as mean±standard

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deviation (SD). The results were analyzed by one-way ANOVA followed by the Tukey post hoc

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test (p < 0.05). Statistical analysis was carried out using Statistical Package for the Social Sciences

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20.0 software and p < 0.05 was considered significant.

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RESULTS

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LC-MS Analysis of CAVAP-W

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The total phenolic content of CAVAP-W was determined to be 3.7%. Fig 1 displayed the LC-

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MS chromatogram and the identification data. Nine compounds including eriocitrin/neoeriocitrin,

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eriocitrin/neoeriocitrin, rhoifolin, hesperidin, naringin, rutin, veronicastroside, neohesperidin and

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hesperetin were identified in CAVAP-W. Specifically, eriocitrin and neoeriocitrin were isomeric

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compounds. Generally speaking, we may not be able to identify and distinguish eriocitrin and

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neoeriocitrin based on the mass spectrum. Naringin and hesperidin were also detected in Korea C.

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aurantium 10 and C. aurantium var. bigaradia 23 according to the published literatures. In fact, all

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the nine compounds were identified for the first time in the analyzed sample of CAVAP-W for the

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fact that there were no published literatures on the study of polyphenols from blossoms of CAVA.

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Inhibitory Effects on Hemolysis and LPS-induced NO Production

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The potential cytotoxicity of CAVAP-W on LO2 and RAW264.7 cells was evaluated by

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MTT assay. The results (data not shown) demonstrated that CAVAP-W displayed no cellular

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toxicity (cell viability all exceeded 90%) at concentrations up to 500 µg/mL. Therefore, 15.625,

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31.25, 62.5, 125, 250 and 500 µg/mL were chosen as working concentrations for further research.

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As shown in Fig 2A, CAVAP-W showed the highest hemolysis inhibition ratio of 58.91% at the

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maximum concentration of 500 µg/mL, close to that of the positive control heparin sodium

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(89.11%) at the same concentration (Fig 2A). Of note, at low concentration of 15.625 µg/mL, the

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hemolysis inhibition activity of CAVAP-W was 63.75 %, much higher than that of the positive

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control heparin sodium (19.7%).

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Furthermore, CAVAP-W significantly suppressed the production of NO at 500 µg/mL and

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the inhibition activity was close to that of the positive control DXM (50 µg/mL) (Fig 2B).

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Additionally, effects of CAVAP-W on morphological changes in LPS-stimulated RAW264.7 cells

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were also evaluated in the current research. As shown in Fig 3, LPS-induced RAW264.7 cells

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increased in size and became irregular in shape, which was in accordance with the large quantities

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of NO in response to inflammatory stimulus of LPS. However, the cells possessed relatively

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smooth surfaces after treated with CAVAP-W at different concentrations of 125, 250 and 500

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µg/mL.

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Anti-inflammatory Activity Assay

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Inhibitory Effect of CAVAP-W on Cytokine Secretion

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In order to further evaluate the anti-inflammatory effect of CAVAP-W, the secretion of

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cytokines including IL-6, TNF-ɑ and IL-1ß were investigated. Obviously, CAVAP-W displayed

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the strongest inhibition effect on expression of IL-6 in a concentration-dependent manner (Fig 4A).

227

For example, the suppression effect of CAVAP-W (125 µg/mL) on expression of IL-6 was greater

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than the positive control DXM (50 µg/mL). At concentrations of 31.25, 125, 250 and 500 µg/mL,

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CAVAP-W showed significant inhibitory effects on TNF-ɑ production (Fig 4B). However, when

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RAW264.7 cells were stimulated with CAVAP-W at 62.5 µg/mL, the accumulation of TNF-ɑ

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(28281.82 pg/mL) even exceeded that of LPS (26781.82 pg/mL), indicating that the concentration

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of CAVAP-W was closely related to the secretion of TNF-ɑ. Simultaneously, CAVAP-W varying

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from 31.25 to 500 µg/mL showed significant inhibition effects on IL-1ß production without a 9



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concentration-dependent manner (Fig 4C). In support, published data also found that polyphenols

235

from blueberries could mediate the balances in pro-inflammatory cytokines of IL-1 and IL-6, thus

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exhibiting anti-inflammatory activity.24

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Inhibitory Effect of CAVAP-W on mRNA Expression of iNOS, IL-6, TNF-ɑ and

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IL-1ß

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The inhibitory effect of CAVAP-W on expression of iNOS on transcription level was

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demonstrated in Fig 4D. The expression of iNOS mRNA was the lowest in normal RAW264.7

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cells while sharply increased in LPS-induced cells. When CAVAP-W was added to the media, the

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expression of iNOS mRNA was significantly suppressed, indicating that CAVAP-W might inhibit

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NO production through suppressing iNOS mRNA expression. Consistently, Hwang et al. also

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reported that NO production was closely associated with the gene expression of iNOS.25

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Consistent with the data in 3.4.1, CAVAP-W also potently inhibited expression of IL-6 mRNA

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(Fig 4E) and IL-1ß mRNA (Fig 4G). The mRNA level of TNF-α was not significantly affected by

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CAVAP-W at 62.5-250 µg/mL. CAVAP-W only suppressed the expression of TNF-ɑ mRNA at

248

its maximum concentration of 500 µg/mL (Fig 4G). According to Fig 4B and F, CAVAP-W

249

showed higher inhibitory effect on TNF-α gene expression than that on TNF-α production. One

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possible explanation would be that TNF-ɑ acted through two transmembrane receptors including

251

TNFR1 and TNFR2.26 Another reason could be that the production of some other pro-

252

inflammatory mediators, such as IL-6 and IL-1ß, could also regulate the secretion of TNF-ɑ from

253

activated macrophages.27-30

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Inhibitory Effect of CAVAP-W on COX-2 Activation

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COX-2 is an inducible enzyme and undetectable in most normal tissues. However, it could be

256

abundantly expressed in activated macrophages stimulated with LPS.31 Therefore, COX-2 could

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serve as target for developing novel therapeutic options. As shown in Fig 4H, CAVAP-W

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produced a concentration-dependent decrease in expression of COX-2 mRNA. Subsequently, this

259

was further confirmed by examining the expression of COX-2 protein using western blot method

260

(Fig 6A-1 and A-2). CAVAP-W at 125-500 µg/mL potently suppressed the expression of COX-2

261

protein in a concentration-dependent manner. Consistently, rheosmin isolated from pine needle

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also significantly decreased COX-2 expression in RAW264.7 cells stimulated with LPS through

263

inhibiting activation of NF-kB pathway.32

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Inhibitory Effect of CAVAP-W on NF-κB Activation

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Immunocytochemical analysis suggested that CAVAP-W dramatically inhibited the

266

translocation of NF-kB subunit P65 into the nucleus in LPS-stimulated macrophages compared

267

with control cells (Fig 5). Consistent with immunocytochemical analysis, western blot analysis

268

further showed that CAVAP-W treatment enhanced the cytosolic P65 (Fig 6C-1 and C-2),

269

resulting in a decrease in the nuclear P65 (Fig 6D-1 and D-2). Also, CAVAP-W dramatically

270

inhibited the phosphorylation of P65 (Fig 6B-1 and B-2). In addition, CAVAP-W significantly

271

inhibited the proteolytic degradation of IκBɑ (Fig 6E-1 and E-2), phosphorylation of IκBɑ (Fig

272

6F-1 and F-2) and phosphorylation of IκKɑ/ß (Fig 6G-1 and G-2). For example, the expression of

273

IκBɑ in RAW264.7 cells was sharply improved when administrated with CAVAP-W at 500

274

µg/mL, close to that in control cells. In support, several phytochemicals showed anti-inflammation

275

potential by inhibiting activation and translocation of NF-kB from the cytoplasm to the nucleus.33-

276

36

277

Inhibitory Effect of CAVAP-W on MAPKs Phosphorylation

278

In order to further investigate the mechanism of CAVAP-W in regulation of inflammatory

279

response, the inhibition effect of CAVAP-W on MAPKs signaling pathways were also detected.

280

As shown in Fig 6E-1 and E-2, CAVAP-W significantly suppressed the phosphorylation of JNK.

281

The expression level of phospho-P38 was down-regulated when administrated with LPS,

282

consistent with published report.21 However, the expression level of phospho-P38 was up-

283

regulated when pre-treated with CAVAP-W at 125-500 µg/mL and was close to that of the control

284

cells without samples (Fig 6F-1 and F-2). These results indicated that MAPKs signaling pathway

285

was also blocked.

286

DISCUSSION

287

The crude polyphenols of CAVA (CAVAP-W) were extracted with distilled water and the

288

yield of polyphenols was determined to be 3.7% with Folin–Ciocalteu reagent. Consistent with our

289

results, Karimi also reported that the total phenolic content of boiling water extracts from Citrus

11



290

aurantium bloom was 3.93%.37 Then LC-MS analysis demonstrated that CAVAP-W was mainly

291

composed of eriocitrin/neoeriocitrin, eriocitrin/neoeriocitrin, rhoifolin, hesperidin, naringin, rutin,

292

veronicastroside, neohesperidin and hesperetin. LC-MS method, a powerful technique for the

293

characterization and structural elucidation in the identification of polyphenols were employed in

294

many published reports.38, 39

295

Furthermore, we investigated the anti-complement and anti-inflammatory effects of CAVAP-

296

W. CAVAP-W displayed comparable hemolysis and NO inhibition rate to the positive control of

297

heparin sodium and DXM, respectively. Complement was one of the major mediators of the

298

inflammatory response. Agents with anti-inflammatory activities were usually found to display

299

significant anti-complement effects. Numerous published data reported that the complement

300

system was closely related to the inflammatory process.40,

301

demonstrated that the anti-inflammatory activity of triterpenes was probably owing to their anti-

302

complementary activity.42 Consistent with published reports,40-42 it could be inferred that the

303

potent inhibitory effect of CAVAP-W on NO production (Fig 2B) might be also closely related to

304

that on hemolysis (Fig 2A). Therefore, further study on CAVAP-W was conducted to reveal the

305

underlying molecular mechanism of its anti-inflammatory effects.

41

Specifically, previous study

306

ELISA and RT-PCR analysis further indicated that CAVAP-W potently inhibited the

307

secretion of IL-6, TNF-α and IL-1β, as well as the mRNA expression of IL-6, TNF-α, IL-1β and

308

COX-2 on transcription levels. A growing number of published reports demonstrated that NF-κB

309

was an important factor in the regulation of iNOS, IL-6, TNF-ɑ and IL-1ß.43, 44 For example, the

310

NF-κB-binding element at -85 bp of murine iNOS promoter played a critical role in iNOS

311

expression and NO production.45 So the immunocytochemical analysis and western blot analysis

312

were performed to investigate whether NF-κB was involved in regulation of inflammatory

313

response of CAVAP-W. The results showed that CAVAP-W significantly blocked NF-κB

314

activation by inhibiting the nuclear translocation of nuclear factor-kappa B (NF-κB) P65,

315

degradation and phosphorylation of IκBα, phosphorylation of IκBα and IκKBɑ/ß. Furthermore,

316

CAVAP-W administration also inhibited MAPK signaling pathway by suppressing

317

phosphorylation of JNK as well as P38. In fact, MAPK signaling pathway was closely associated

318

with NF-κB pathway and they worked together to regulate the inflammatory responses. For 12


Page 12 of 24

Page 13 of 24


319

example, polyozellin isolated from Polyozellus multiplex dramatically inhibited NO accumulation

320

and iNOS gene expression by suppressing activation of NF-κB and MAPK signaling pathways.36

321

Numerous bioactivity investigations on polyphenols extracted from natural products using cell

322

lines, at concentrations in the low-µM-to-mM range, showed that most of these active ingredients

323

appear as phase II metabolites in the circulatory system after dietary intake, and their presence in

324

plasma after dietary intake rarely exceeds nM concentrations.46 However, there were also a

325

growing number of published data demonstrating that modest long-term intakes of polyphenols

326

are beneficial to the treatment of various diseases.47, 48 Furthermore, various methods such as

327

physical activity49 and enzymatic modification50 were reported to be employed to improve the

328

bioavailability of polyphenols. Specifically, Nielsen et al. proved that the bioavailability of

329

hesperidin could be increased by changing the absorption site, which was achieved via enzymatic

330

modification to hesperetin-7-glucoside.50 Therefore, new methods could be used in future

331

interventions to ensure or improve the bioavailability of polyphenols thereby fully exploiting their

332

beneficial properties. The future work will be focused on the purification and characterization of

333

CAVAP-W in order to find the specific compounds responsible for its bioactivities.

334

Funding

335

This research was supported by Science and Technology Project of Guangzhou City

336

(201604020150), Science and Technology Project of Guangdong (2014A020212682), Special

337

Research of Traditional Chinese Medicine Hospital of Guangdong Province (YN2014ZH02).

338

Notes

339

The authors declare no competing financial interest.

340

REFERENCES

341 342 343 344 345 346 347 348 349

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498 499

16


Page 16 of 24

Page 17 of 24


500 501 502

Figure legends

503

5, 6, 7, 8 and 9 represented neohespeidoside (C27H32O15), neoeriocitrin (C27H32O15), rhoifolin

504

(C27H30O14), hesperidin (C28H34O15), naringin (C27H32O14), rutin (C27H30O16), veronicastroside

505

(C27H30O15), neohesperidin (C29H38O23) and hesperetin (C16H14O6), respectively. Their fragmens

506

at (M-H)- (m/z) were 595.1708, 595.1708, 577.1583, 609.1851, 579.1743, 609.1491,

507

593.1535, 753.2283 and 301.0721.

508

Fig. 2. Inhibitory effects of CAVAP-W on the classical pathway of the complement system (A);

509

Inhibition effect of CAVAP-W on NO production in LPS-induced RAW264.7 cells (B). All

510

experiments were run in triplicate, and data showed mean ± SD values. *p < 0.05 and **p < 0.01

511

compared to control group.

512

Fig. 3. Pictures of RAW264.7 cells by an invert microscope after treated with CAVAP-W at

513

different concentrations.

514

Fig. 4. Effects of CAVAP-W on LPS-induced secretion of IL-6 (A), TNF-α (B) and IL-1ß (C);

515

Effects of CAVAP-W on LPS-induced mRNA expression of iNOS (D), IL-6 (E), TNF-α (F), IL-

516

1ß (G) and COX-2 (H). All experiments were run in triplicate, and data showed mean ± SD values.

517

*p < 0.05 and **p < 0.01 compared to LPS-treated group, while

518

control group.

519

Fig. 5. Analysis of nuclear translocation of NF-κB P65 of CAVAP-W by immunofluorescence

520

analysis.

521

Fig. 6. Effects of CAVAP-W on the COX-2 (A-1, A-2); phospho-P65 (B-1, B-2); cytosol P65 (C-

522

1, C-2); nucleus P65 (D-1; D-2); IκBα (E-1, E-2); phospho-IκBα (F-1, F-2); phospho-IκKα/ß (G-1,

523

G-2); phospho-JNK (H-1, H-2); phospho-P38 (I-1, I-2);. All experiments were run in triplicate,

524

and data showed mean ± SD values. *p < 0.05 and **p < 0.01 compared to LPS-treated group,

525

while ##p < 0.01 compared to the control group.

Fig 1. MS chromatogram of CAVAP-W. The numbers represents the compounds.. Peaks 1, 2, 3, 4,

526

17


##

p < 0.01 compared to the


527 528

Fig 1. The total ion chromatogram of CAVAP-W. Peaks 1, 2, 3, 4, 5, 6, 7, 8 and 9 represented

529

eriocitrin/neoeriocitrin (C27H32O15), eriocitrin/neoeriocitrin (C27H32O15), rhoifolin (C27H30O14),

530

hesperidin (C28H34O15), naringin (C27H32O14), rutin (C27H30O16), veronicastroside (C27H30O15),

531

neohesperidin (C29H38O23) and hesperetin (C16H14O6), respectively. Their precursor ions [M-H]-

532

of each individual were at m/z 595.1708, 595.1708, 577.1583, 609.1851, 579.1743, 609.1491,

533

593.1535, 753.2283 and 301.0721, respectively.

534

18


Page 18 of 24

Page 19 of 24


535 CAVAP-W Heparin sodium

80 60

∗∗

∗∗

100

∗∗

NO inhibition (%)

Hemolysis inhibition (%)

100

∗∗ ∗∗ ∗

∗

∗∗

∗

∗∗

40 20

∗∗

∗

15.625 31.25 62.5 125 250 500 concentration (µg/mL) A

80

∗∗ ∗∗

60 40 ∗

20

∗∗

0 DXM 15.625 31.25 62.5 125 250 500 CAVAP-W (µg/mL) B

536

Fig. 2. Inhibitory effects of CAVAP-W on the classical pathway of the complement system (A);

537

Inhibition effect of CAVAP-W on NO production in LPS-induced RAW264.7 cells (B). All

538

experiments were run in triplicate, and data showed mean ± SD values. *p < 0.05 and **p < 0.01

539

compared to control group.

540

19



541 542

Fig. 3. Pictures of RAW264.7 cells by an invert microscope after treated with CAVAP-W at

543

different concentrations.

544

20


Page 20 of 24

Page 21 of 24


∗∗ ∗∗

∗∗

∗∗ ∗∗

∗∗

Con LPS DXM 31.25 62.5 125 250 500 CAVAP-W (µg/mL) A

35 30 25 20 15 10 5 0

TNF-α accumulation (pg/mL)

##

35000 30000 25000 20000 15000 10000 5000 0

iNOS mRNA expression

3500 3000 2500 2000 1500 1000 500 0

IL-1β accumulation (pg/mL)

IL-6 accumulation (pg/mL)

545

## ∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

Con LPS DXM 31.25 62.5 125 250 500 CAVAP-W (µg/mL) C

30000 ∗∗ ∗∗

∗∗

∗∗

10000 0

∗∗

Con LPS DXM 62.5 125 250 500 CAVAP-W (µg/mL) E

COX-2 mRNA expression

IL-1β mRNA expression

##

100000 80000

∗∗

∗∗

## ∗∗

∗∗

∗∗

∗∗ ∗∗

Con LPS DXM 62.5 125 250 500 CAVAP-W (µ g/mL ) D ## ∗∗ ∗∗

## ∗∗

3000

∗∗

∗∗

60000

∗∗

2000

40000

∗∗

∗∗

∗∗

1000

20000 0

Con LPS DXM 31.25 62.5 125 250 500 CAVAP-W (µg/mL) B

4000

∗∗

∗∗

∗∗

C o n L P S D X M 6 2 .5 1 2 5 2 5 0 5 0 0 C A V A P -W ( µ g /m L ) F 5000

140000 120000

∗∗

TNF-α mRNA expression

IL-6 mRNA expression

##

20000

∗∗ ∗∗

14 12 10 8 6 4 2 0

50000 40000

7000 6000 5000 4000 3000 2000 1000 0

##

C on LPS D X M 62.5 125 250 500 CA VA P-W ( µ g/mL)

0 C on

LPS D X M 62.5 125 250 500

546

C A V A P-W (µ g/m L ) H G Fig. 4. Effects of CAVAP-W on LPS-induced secretion of IL-6 (A), TNF-α (B) and IL-1ß (C);

547

Effects of CAVAP-W on LPS-induced mRNA expression of iNOS (D), IL-6 (E), TNF-α (F), IL-

548

1ß (G) and COX-2 (H). All experiments were run in triplicate, and data showed mean ± SD values.

549

*p < 0.05 and **p < 0.01 compared to LPS-treated group, while

550

control group.

551

21


##

p < 0.01 compared to the


552 553

Fig. 5. Analysis of nuclear translocation of NF-κB P65 of CAVAP-W by immunofluorescence

554

analysis.

22


Page 22 of 24

Page 23 of 24


555

B-1

0.3

∗∗

∗∗

0.2

0.4 ∗∗

0.2

∗∗

0.1 0.0

0.0 Con LPS 125 250 500 CAVAP-W (µg/mL) A-2

Con LPS 125 250 500 CAVAP-W (µg/mL)

Relative density of IκBα

∗∗ ∗∗

∗∗

0.4

∗∗

0.0

Con LPS 125 250 500 CAVAP-W (µg/mL) D-2

Con LPS 125 250 500 CAVAP-W (µg/mL) E-2

Relative density of p-IκKα/β

Relative density of p-JNK

##

∗∗ ∗∗

0.4

∗∗

∗∗

0.2

0.2 0.0 Con LPS 125 250 500 CAVAP-W (µ g/mL )

Con LPS 125 250 500 CAVAP-W (µg/mL)C-2

F-1 ##

∗∗ ∗∗ ∗∗

0.0 Con LPS 125 250 500 CAVAP-W (µg/mL) F-2

I-1

H-1 0.6

0.4

0.0

0.2

0.8

∗∗

0.2

0.4

G-1 ##

∗∗

0.6

##

0.1

0.2

0.6

∗∗

0.8

∗∗

0.2

∗∗

0.8

∗∗ ##

0.4

1.0

0.3

##

0.6

0.6

B-2

0.4

0.8

0.8

E-1

D-1 1.0 Relative density of P65

Relative density of P65

∗∗

Relative density of p-IκBα

0.6

##

0.4

##

0.0 C on LPS 125 250 500 C A V A P-W (ug/mL)

Relative density of p-P38

0.8

0.0

C-1 1.0

0.5

Relative density of p-P65

Relative density of COX-2

A-1 1.0

0.3 ∗∗ ∗∗

0.2

∗∗ ##

0.1 0.0

Con LPS 125 250 500

556

CA V AP-W (µ g/mL ) I-2 H-2 G-2 Fig. 6. Effects of CAVAP-W on the COX-2 (A-1, A-2); phospho-P65 (B-1, B-2); cytosol P65 (C-

557

1, C-2); nucleus P65 (D-1; D-2); IκBα (E-1, E-2); phospho-IκBα (F-1, F-2); phospho-IκKα/ß (G-1,

558

G-2); phospho-JNK (H-1, H-2); phospho-P38 (I-1, I-2). All experiments were run in triplicate, and

559

data showed mean ± SD values. *p < 0.05 and **p < 0.01 compared to LPS-treated group, while

560

##

p < 0.01 compared to the control group.

561 23



562

TOC graphic

563

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Polyphenols from Blossoms of Citrus aurantium ... - ACS Publications

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