Antiallergic Activity of Ethanol Extracts of Arctium lappa L. Undried

Apr 18, 2016 - ... Health Sciences and Technology, Inje University, Busan 47392, Republic of Korea .... Dried and undried rhizomes of A. lappa (each 1...
0 downloads 0 Views 2MB Size
Subscriber access provided by UOW Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

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

2

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

3

2H3 cells

4

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

5

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

6

1

7

Republic of Korea

8

2

9

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

10

3

11

University, Yongin-si, Republic of Korea

12

4

13

Korea

14

5

15

Republic of Korea

16

*Corresponding authors:

17

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

18

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

19

mail: [email protected]

20

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

21

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

22

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

23



24

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.

25 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

26

Abstract

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39 40 41 42

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

43

2

ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

Journal of Agricultural and Food Chemistry

44

Introduction

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

problems3, 4.

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

69

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

70

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

71

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

72

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

73

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

74

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

75

are representative markers of the allergic inflammatory reactions.

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

induced sleep18 and suppressed the inflammatory response in lipopolysaccharide-stimulated

89

murine BV2 microglial cells19.

90

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

91

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

92

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

93

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

ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38

Journal of Agricultural and Food Chemistry

94

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

95

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

96

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

97

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

98

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

99

MAPK.

100 101

Materials and methods

102

Chemicals and reagents

103

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

104

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

105

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

106

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

107

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

108

Fractionation and isolation of A. lappa

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

Experimental animals

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

Passive cutaneous anaphylaxis (PCA) assay in rats

140

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

141

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

142

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

143

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

ACS Paragon Plus Environment

Page 6 of 38

Page 7 of 38

Journal of Agricultural and Food Chemistry

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

Compound

153

measurement in mice

48/80-induced

systemic

anaphylaxis

assay

and

serum

histamine

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

spectrofluorometric procedure24. The fluorescent intensity was measured at 439 nm

163

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

164

β-Hexosaminidase release assay

165

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

166

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

167

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

168

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

Cell viability assay

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

survival.

190

Histamine release assay in RBL-2H3 cells

191

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

192

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

193

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

ACS Paragon Plus Environment

Page 8 of 38

Page 9 of 38

Journal of Agricultural and Food Chemistry

194

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

195

(Cayman Chemical, Michigan, USA).

196

Measurement of TNF- α and IL-4 release

197

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

198

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

199

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

200

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

201

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

202

USA).

203

Western blotting

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

219

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

220

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

221

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

222

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

223

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

224

Statistical analysis

225

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

226

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

227

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

228

Chicago, IL, USA).

229 230

Results

231

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

232

hexosaminidase release and cell viability

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

ACS Paragon Plus Environment

Page 10 of 38

Page 11 of 38

Journal of Agricultural and Food Chemistry

244

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

245

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

246

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

247

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

248

Inhibition of passive cutaneous anaphylaxis (PCA) by ALE treatments

249

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

250

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

251

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

252

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

253

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

254

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

255

histamine release in mice

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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