Antiallergic Activity of Ethanol Extracts of Arctium lappa L. Undried

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Anti-allergic activity of ethanol extracts of Arctium lappa L. undried roots and its active compound–oleamide–in regulating Fc#RI-mediated and MAPK signaling in RBL-2H3 cells Woong-Suk Yang, Sung Ryul Lee, Yong Joon Jeong, Dae Won Park, Young Mi Cho, Hae Mi Joo, Inhye Kim, Young-Bae Seu, Eun-Hwa Sohn, and Se Chan Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00425 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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Journal of Agricultural and Food Chemistry

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Anti-allergic activity of ethanol extracts of Arctium lappa L. undried roots and its active

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compound––oleamide––in regulating FcεRI-mediated and MAPK signaling in RBL-

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2H3 cells

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Woong-Suk Yang1¶, Sung Ryul Lee2¶, Yong Joon Jeong3, Dae Won Park3, Young Mi Cho3,

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Hae Mi Joo4, Inhye Kim3, Young-Bae Seu1, Eun-Hwa Sohn5*, and Se Chan Kang3*

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1

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Republic of Korea

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Technology, Inje University, Busan, Republic of Korea

School of Life Sciences and Biotechnology, Kyungpook National University, Daegu,

Cardiovascular and Metabolic Disease Center and Department of Health Sciences and

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3

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University, Yongin-si, Republic of Korea

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4

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Korea

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Republic of Korea

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*Corresponding authors:

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Dr. Eun-Hwa Sohn,, Department of Herbal Medicine Resource, Kangwon National

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University, Samcheok-si, Kangwon-do, Republic of Korea, 25913, Tel +83 33 540 3322, E-

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mail: [email protected]

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Dr. Se Chan Kang, Department of Oriental Medicine Biotechnology, College of Life

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Sciences, Kyung Hee University, Yongin-si, Gyeonggi-do, Republic of Korea, 17104, Tel

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+82 31 201 2687, Fax +82 31 204 8116, E-mail: [email protected]

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¶

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Short tile: Anti-allergic activity of Arctium lappa L. and oleamide

Department of Oriental Medicine Biotechnology, College of Life Sciences, Kyung Hee

Radiation Health Research Institute, Korea Hydro & Nuclear Power Co., Ltd., Republic of

Department of Herbal Medicine Resource, Kangwon National University, Samcheok,

These authors contributed equally to this work.

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ACS Paragon Plus Environment


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Abstract

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The anti-allergic potential of Arctium lappa L. was investigated in Sprague-Dawley rats, ICR

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mice, and RBL-2H3 cells. Ethanol extract (90%) of A. lappa (ALE, 100 µg/ml) inhibited the

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degranulation rate by 52.9%, determined by the level of β-hexosaminidase. ALE suppressed

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passive cutaneous anaphylaxis (PCA) in rats and attenuated anaphylaxis and histamine

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release in mice. To identify the active compound of ALE, we subsequently fractionated and

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determined the level of β-hexosaminidase in all sub-fractions. Oleamide was identified as an

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active compound of ALE, which attenuated the secretion of histamine and the production of

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tumor necrosis factor (TNF)-α and interleukin-4 (IL-4) in cells treated with compound 48/80

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or A23187/PMA. Oleamide suppressed FcεRI-tyrosine kinase Lyn-mediated pathway, c-Jun

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N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinases (p38-MAPKs).

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These results showed that ALE and oleamide attenuated allergic reactions and should serve

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as a platform to search for compounds with anti-allergic activity.

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Key words; Arctium lappa L, oleamide, anti-allergic, Lyn, RBL-2H3 cell

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Introduction

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Allergy is a hypersensitivity disorder of the immune system1. Allergic rhinitis, asthma, and

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atopic eczema are the most common causes of chronic ill-health. The increased prevalence of

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atopic disease in Western Europe, USA, Australasia, and Asia has considerably burdened the

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quality of life and health-care costs, even in developed countries1. In several etiological

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studies, it was suggested that the widespread use of antibiotics for minor illness in early life1

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in "westernized life-style", and also other environmental or genetic situation may change

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immunity from type 1 T helper (Th1) to type 2 T helper (Th2), which can induce excessive

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immunoglobulin E (IgE) production and lead to an allergic response2. The cytokines released

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by Th2 cells induce the proliferation and activation of mast cells and eosinophils in allergic

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inflammation, leading to impaired function of the skin barrier and more serious skin

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problems3, 4.

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Mast cells are key effector cells in IgE-associated immune responses at the early and late

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phases of allergic reactions5. Mast cells are activated by allergen and IgE, they undergo IgE-

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dependent maturation, and secrete allergic related mediators such as histamine, β-

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hexosaminidase, and tryptase. These mediators are pre-formed and stored in granules, but are

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also newly synthesized upon activation. Antigenic cross-linking of the IgE receptor Fc

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epsilon RI (FcεRI) on mast cells result in immediate histamine secretion (within minutes),

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and induce prostaglandins, leukotrienes, and inflammatory cytokines such as TNF-α, IL-4,

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and IL-5, which play pivotal roles at the late phase of an allergic reaction6.

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FcεRI cross-linking activates Src tyrosine kinase families, such as tyrosine-protein kinase

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Lyn (Lyn), spleen tyrosine kinase (Syk), and Src family tyrosine kinase (Fyn), which

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regulates mast cell functions7. As an intermediator or effector of FcεRI-Src kinases signaling,

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phospholipase C (PLC), protein kinase C (PKC), c-Jun N-terminal kinases (JNK/SAPK), p38

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mitogen-activated protein kinases (p38-MAPKs), and other kinases/phosphatases exert 3



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allergic response through degranulation or cytokine gene expression of mast cells7.

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Mast cells are major sources of TNF-α and IL-4, which play a pivotal role in allergic

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reactions such as skin inflammation, hypersensitivity, and itching6. IL-4 stimulates the

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production of IgE in B cells and is associated with anaphylaxis. In addition, IL-4 contributes

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to the expansion of Th2 cell subset from naive T cells and isotype switching of B cells to

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produce IgE against specific environmental allergens8. Cytokines, such as TNF-α and IL-4,

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are representative markers of the allergic inflammatory reactions.

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Arctium lappa L. is known as burdock and is a popular edible vegetable cultivated in Asia

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and Europe. The dried roots are widely used as a food ingredient or as a major source of

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crude drug for different therapeutic purposes9. Several reports have documented that their

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seeds are used in traditional Korean medicine as diuretic, anti-inflammatory or detoxifying

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agents10, 11. The major bioactive ingredients isolated from A. lappa are arctigenin, arctiin,

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trachelogenin, lappaol F, diarctigenin, β-eudesmol, caffeic acid, chlorogenic acid, tannin,

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inulin, and sitosterol-β-D-glucopyranoside11-14.

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We as well as others have previously proposed the anti-allergic effects of A. lappa extract,

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however the extract with n-butanol is not suitable for clinical use. Moreover, the bioactive

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components involved in anti-allergic effects were not clarified15, 16.

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Oleamide (cis-9-octadecenamide), an amide of oleic acid, was isolated from the

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cerebrospinal fluid of sleep-deprived cats17. Previous studies demonstrated that oleamide

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induced sleep18 and suppressed the inflammatory response in lipopolysaccharide-stimulated

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murine BV2 microglial cells19.

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In order to examine the anti-allergic effects of Arctium lappa L. extract for clinical use, we

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provided ethanol extract of Arctium lappa L. to animals and investigated the local and

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systemic allergic responses. To identify the bioactive compound of A. lappa, which has anti-

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allergic activity, we subsequently performed further fractionation and all sub-fractions were 4


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subjected to measurement of their β-hexosaminidase and histamine releases in RBL-2H3

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mast cells. This approach led to the identification of the major bioactive component,

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oleamide, and provided novel insight into its anti-allergic effect. To identify the possible

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mechanism of oleamide, we investigated FcεRI-mediated signaling events, Fyn, Syk, PKC,

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PLCγ, and MAPKs including JNK, extracellular signal-regulated kinase (ERK) and p38

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MAPK.

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Materials and methods

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Chemicals and reagents

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Fetal bovine serum (FBS), penicillin/streptomycin (P/S), and minimum essential medium

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(MEM) were obtained from Invitrogen (CA, USA). Dinitrophenyl-specific IgE (DNP-IgE),

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DNP-BSA, 4-Nitrophenyl N-acetyl-b-D-glucosaminide (NP-GlcNAc), and piperazine-N, N′-

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bis (2-ethanesulfonic acid) [PIPES] were purchased from Sigma-Aldrich (MO, USA). Unless

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indicated otherwise, chemicals were purchased from Sigma (MO, USA).

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Fractionation and isolation of A. lappa

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Rhizomes of Arctium lappa L. were collected from Jecheon (ChungBuk, Korea) in

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October 2012, and identified by Prof. Kang Se Chan, Kyung Hee University (Yongin, Korea).

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The voucher specimen (NMR114) was deposited in the Laboratory of Natural Medicine

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Resources, Kyung Hee University. Dried and undried rhizomes of A. lappa (each 1 kg) were

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extracted with 50% ethanol (EtOH), and 112 g and 164 g of each extract, respectively were

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obtained. Among them, undried rhizomes of A. lappa (500 g) were extracted with 90%

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ethanol under reflux (5 L, 24 h). The solutions of extract were filtered, and then evaporated at

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40°C under reduced pressure, yielding 45 g of dry powder. This crude extract was stored at -

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20°C until further use. Approximately 90% EtOH extract (ALE) was suspended in 1 L of

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distilled water and sequentially partitioned with equal volumes of n-hexane, CH2Cl2, EtOAc, 5



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and n-BuOH for fractions of n-hexane, CH2Cl2, EtOAc, n-BuOH, and H2O (Scheme 1). A

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portion of the CH2Cl2 fraction (ALME, 3.7 g) was chromatographed on silica gel (11.5 × 58

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cm, 2 kg), using a graded series of CH2Cl2 and MeOH (98:2 ~1:1 for gradient) as the eluent

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to yield five fractions (ALM1~5) based on their polarities. Fraction ALM1 was

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chromatographed on a silica gel column (3.8 × 62 cm, 300 g), eluted with n-hexane : EtOAc

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(4:1 ~1:1) to yield three subfractions (SAI-1~3). Fraction SAI-2 was rechromatographed with

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high-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan), eluted with

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MeOH : H2O (75:25 ~ only MeOH) to yield six subfractions (SAI-2-1~6). SAI-2-3

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subfraction was recrystallized with MeOH for the isolation of pure oleamide (13.8 mg)

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(Scheme 2). The chemical structure of oleamide was verified by comparing the NMR data to

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those of previously reported studies20 and the reference oleamide obtained from Sigma.

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Experimental animals

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Sprague-Dawley male rats (SD rats, 6 weeks old, 200–230 g) and ICR male mice (6 weeks

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old, 20–22 g) were purchased from Central Lab, Animal Inc. (Seoul, Korea) and housed in an

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animal room, maintained at temperature of 23 ± 1°C and humidity of 55 ± 5%, with 12-h

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light/12-h dark cycle. The rats and mice were fed a standard laboratory diet with tap water ad

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libitum. All experimental procedures were performed in compliance with the NIH Guide for

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the Care and Use of Laboratory Animals and National Animal Welfare Law in Korea. The

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experimental animal facility and protocols were approved by the Institutional Animal Care

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and Use Committee of Semyung University (SMU-2011-008).

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Passive cutaneous anaphylaxis (PCA) assay in rats

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Passive cutaneous anaphylaxis (PCA) is one of the simplest allergic cutaneous reactions in

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vivo and is widely used for evaluation of anti-allergic compounds21. Activated mast cells by

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passive sensitization with antigen-specific IgE release vasoactive mediators and increase

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vascular permeability immediately after antigen challenge is visualized by extravasation of 6


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the dye given simultaneously with the antigen. Rats were injected intravenously with 250 µg

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antigen (Ag, DNP-BSA) in 250 µL PBS containing 4% evans blue 24 h after intradermal

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administration of a DNP-specific IgE (0.5 µg) into the ear. To measure the activity of ALE

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(25, 50, and 100 mg/kg), it was administered orally 1 h before DNP-BSA administration. The

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rats were euthanized 1 h after treatment with the Ag and the treated ear was excused in order

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to measure the amount of dye extravasated. The dye was extracted from the ear with 700 µL

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formamide at 63°C overnight, as previously described22. The absorbance at 620 nm was

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measured with a Multi-reader (TECAN, Infinite 200, Zürich, Switzerland).

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Compound

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measurement in mice

48/80-induced

systemic

anaphylaxis

assay

and

serum

histamine

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Compound 48/80 is a mixed polymer of phenethylamine, cross-linked by formaldehyde

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and has been used as a direct and convenient agent for the study of unknown compounds on

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mast cell-dependent anaphylactic reaction23. Mice were injected intraperitoneally (i.p.) 8

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mg/kg of compound 48/80. ALE were dissolved in saline and administered orally (25, 50 and

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100 mg/kg) 1 h before the injection of compound 48/80 (n = 6/group). Mortality was

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monitored for 1 h after the induction of anaphylactic shock. After the mortality test, blood

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was obtained from the heart of each mouse. The blood was centrifuged at 400 g for 10 min.

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The plasma was withdrawn and histamine content was measured by the o-phthalaldehyde

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spectrofluorometric procedure24. The fluorescent intensity was measured at 439 nm

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(excitation at 353 nm) using a spectrofluorometer (Shimadzu, RF-5301 PC, Kyoto, Japan).

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β-Hexosaminidase release assay

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RBL-2H3 rat mast cell lines were obtained from the American Type Culture Collection

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(ATCC, Rockville, MD, USA). The level of β-hexosaminidase release from RBL-2H3 cells

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were measured using a modified version of a previously-described method25. Briefly, RBL-

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2H3 cells (1 × 104 cells/well) were plated in a 96-well plate. The cells were sensitized with 7



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anti-DNP-IgE (100 ng/mL) for 16 h at 37°C. After washing the cells with PIPES buffer (25

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mM PIPES, pH 7.2; 119 mM NaCl, 5mM KCl, 0.4 mM MgCl2 ·6H2O, 1 mM CaCl2, 5.6 mM

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glucose, 40 mM NaOH and 0.1% Bovine serum albumin), they were pretreated with various

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doses of sample for 30 min and then treated with DNP-BSA (1 µg/mL) for 30 min at 37°C.

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Aliquots of the cellular supernatant (15 µL) were transferred to a 96-well plate and incubated

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with 60 µL of substrate (1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.05 M citrate,

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pH 4.5) for 60 min at 37°C. The stop buffer (150 µL, 0.1 M Na2CO3-NaHCO3 buffer, pH 10)

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was added to stop the reaction. The absorbance was measured at 405 nm using a Multi-reader

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(TECAN, Infinite 200, Zürich, Switzerland). Ketotifen, a second-generation non-competitive

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H1-antihistamine and mast cell stabilizer, was used as the positive control.

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Cell viability assay

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To evaluate viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

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assay was performed as described previously with slight modification21. RBL-2H3 cells were

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grown in MEM with 15% FBS and 2 mM L-glutamine at 37°C in a humidified incubator

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with 5 % CO2. Briefly, RBL-2H3 cells (1×104 cells/well) were seeded in 96-well plates and

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incubated overnight. The cells were then treated with various doses of samples for 24 h. Ten

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micro liter of 5 mg/mL MTT solution was added to each well and incubated for 4 h. The

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formazan crystals were dissolved in DMSO and the absorbance at 540 nm was determined

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with a Multi-reader (TECAN, Infinite 200, Zürich, Switzerland). Absorbance values were

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normalized to the values obtained for the untreated cells to determine the percentage of

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survival.

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Histamine release assay in RBL-2H3 cells

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RBL-2H3 cells (1×104 cells/well) were seeded in a 96-well plate and then incubated

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overnight in a complete medium. Next, the cells were treated for 1 h with the indicated

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concentrations of samples prior to stimulation with compound 48/80 (10 µg/mL) at 37°C for 8


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20 min. Finally, histamine contents were measured using the histamine enzyme assay kit

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(Cayman Chemical, Michigan, USA).

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Measurement of TNF- α and IL-4 release

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The inhibitory effects on calcium ionophore A23187 (1µM) and PMA (50 nM)-stimulated

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release of cytokines were evaluated in RBL-2H3 cells. Briefly, cells were treated with various

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concentration of oleamide and stimulated with A23187 and PMA for 5 h. The concentrations

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of interleukin-4 (IL-4) and tumor necrosis factor (TNF)-α in the cellular supernatants were

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determined using a commercial enzyme-linked immunoassay (ELISA) kit (eBioscience, CA,

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USA).

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Western blotting

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RBL-2H3 cells were sensitized with anti-DNP-IgE (100 ng/mL) for 16 h at 37°C. After

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washing the cells with PIPES buffer, cells were pretreated with various doses of oleamide for

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30 min and then treated with DNP-BSA (1 µg/mL) for 30 min at 37°C. The cells were

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washed twice with ice-cold PBS and then lysed in a Protein Extraction solution (iNtRON,

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Gyeonggi, Korea), on ice for 1 h. The lysates were clarified by centrifugation at 10,000 g for

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30 min at 4°C. The protein concentrations were determined using a bicinchoninic acid assay

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(BCA) protein Assay kit (Bio-Rad, CA, USA) as previously described26. The samples were

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subjected to sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) using 10% running gels.

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The proteins were transferred onto a nitrocellulose membrane (Bio-Rad, CA, USA), which

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was blocked with 5% non-fat milk/TBST for 1 h at room temperature and subsequently

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incubated overnight at 4°C with primary antibody (diluted 1:1000 in 5% non-fat milk/TBST).

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The membrane was incubated with a secondary antibody for 1 h. The final detection was

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performed with EZ-capture software. Polyclonal anti-phospho-Lyn (sc-139680), polyclonal

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anti-p38 (sc-535), monoclonal anti-phospho-p38 (sc-7973), monoclonal anti-β-actin (sc-

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47778) antibodies, and secondary antibodies (rabbit anti-goat IgG-HRP, sc-2768 and mouse 9



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anti-goat IgG-HRP, sc-2354) were obtained from Santa Cruz (Texas, USA). Polyclonal anti-

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JNK (#9252), monoclonal anti-phospho-JNK (#9255), polyclonal anti-ERK (#9102),

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monoclonal anti-phospho-ERK (#4370), monoclonal anti-phospho-Syk (#2710), polyclonal-

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phospho-PKCα/β (#9375), polyclonal anti-phospho-PKCδ (#2055), and polyclonal anti-

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phospho-PLCγ (#2822) were purchased from Cell Signaling Technology Inc. (MA, USA).

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Statistical analysis

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Results are expressed as means ± standard deviation (SD). Student’s t-test or One-way

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ANOVA/Dunnett’s t-test was used to assess the significance between control groups and

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treated groups. Statistical analysis was performed using SPSS, version 12 (SPSS Inc.,

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Chicago, IL, USA).

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Results

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Comparative study for the effects of dried and undried roots of A. lappa on β-

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hexosaminidase release and cell viability

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The amount of active constituents in plants can be different depending on the status of the

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plant, thus we compared dried and undried A. lappa roots (50% ethanol extracts). Extracts of

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dried and undried roots did not lead to cytotoxicity at treatments of 100 µg/mL (dried: 97.5 ±

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1.34%, undried: 98.1 ± 1.64%, non-treated control: 100.0 ± 2.61%). The release of β-

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hexosaminidase was determined in activated RBL-3H2 cells using anti-DNP-IgE (100

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ng/mL) and DNP-BSA (1 µg/mL). Extract of undried A. lappa roots inhibited β-

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hexosaminidase release (38.4 ± 2.43%) whereas that of dried A. lappa roots inhibited 32.3 ±

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2.06% at 100 µg/mL. We found a higher inhibition rate of β-hexosaminidase release in

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undried A. lappa roots compared to the rate in dried A. lappa roots at 1 and 10 µg/mL.

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To clarify the better extraction condition of A. lappa, different concentrations of EtOH (30,

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70 and 90%) were investigated. The maximal inhibitory effect on β-hexosaminidase release 10


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and the highest viability was shown in the 90% EtOH-fraction of undried A. lappa (52.9 ±

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1.38% and 96.7 ± 0.82%, respectively, compared to those of the control when treated at 100

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µg/mL) (Table 1). Based on these findings we examined the following with 90% EtOH

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extract of the undried root of A. lappa (ALE).

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Inhibition of passive cutaneous anaphylaxis (PCA) by ALE treatments

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The inhibitory effect of ALE on allergic reaction was further confirmed in vivo through

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PCA assay, a typical animal model of IgE-mediated immediate types of allergic reactions.

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The intensity of PCA was determined by extravasation of the injected Evans blue dye. PCA

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responses in rats administered with ALE were significantly reduced in a dose-dependent

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manner (p < 0.05, Fig. 1A).

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Inhibitory effect of ALE on compound 48/80-induced systemic anaphylaxis and serum

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histamine release in mice

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We also examined the inhibitory effects of ALE on compound 48/80-induced systemic

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fatal anaphylaxis in vivo. The mortality was determined by intraperitoneal injection of

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compound 48/80 (8 mg/kg body weight). The control group (200 µL saline) showed 100%

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mortality. Although pre-administration of 25 mg/kg ALE was not effective, 50 mg/kg and

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100 mg/kg of ALE reduced lethality to 83.3% and 66.6%, respectively (Fig. 1B).

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Administration of ALE suppressed the plasma level of histamine evoked by compound 48/80

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insult in a dose-dependent manner. In particular, mice administered ALE 100 mg/kg

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effectively inhibited the release of histamine compared to that of the control group (p

Antiallergic Activity of Ethanol Extracts of Arctium lappa L. Undried

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