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Inhibition of Mast cell-mediated Allergic Responses by Arctii Fructus Extracts and its Main Compound Arctigenin Ji-Ye Kee, and Seung-Heon Hong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02965 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017
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Inhibition of Mast cell-mediated Allergic Responses by Arctii Fructus Extracts and its Main
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Compound Arctigenin
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Ji-Ye Kee, Seung-Heon Hong
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Department of Oriental Pharmacy, College of Pharmacy, Wonkwang-Oriental Medicines
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Research Institute, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 54538, Republic
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of Korea
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Correspondence should be addressed to Seung-Heon Hong,
[email protected] 11
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Short title: Anti-allergic effects of Arctii Fructus and arctigenin
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ABSTRACT: The Arctium lappa (Arctii Fructus) seed and its major active compound,
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arctigenin (ARC), are known to have anti-cancer, anti-obesity, anti-osteoporosis, and anti-
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inflammatory activities. However, the effect of Arctii Fructus and ARC on mast cell-mediated
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allergic inflammation and the associated mechanism have not been elucidated. Therefore, we
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attempted to investigate the anti-allergic activity of Arctii Fructus and ARC on mast cells and
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experimental mouse models. Arctii Fructus water (AFW) or ethanol extract (AFE) and ARC
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reduced the production of histamine and pro-inflammatory cytokines such as interleukin (IL)-
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1β, IL-6, IL-8, and TNF-α in mast cells. AFW, AFE, and ARC inhibited phosphorylation of
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MAPKs and NF-κB in activated mast cells. Moreover, IgE-mediated passive cutaneous
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anaphylaxis and compound 48/80-induced anaphylactic shock were suppressed by AFW, AFE,
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and ARC administration. These results suggest that Arctii Fructus and ARC are potential
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therapeutic agents against allergic inflammatory diseases.
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KEYWORDS: Arctii Fructus, Arctigenin, Allergic inflammation, Mast cells, MAPK
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INTRODUCTION
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Allergic diseases, such as allergic rhinitis, allergic eczema, and atopic dermatitis, are
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hypersensitivity disorders of the immune system. The number of allergy patients has
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consistently increased with approximately 10–20% of the world’s population suffering from
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allergic diseases.1 Thus, allergic diseases are important public health problems that can
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influence the quality of life and the socioeconomic burden. Antihistamines, steroids, non-
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steroidal anti-inflammatory drugs, and immunosuppressants are common drugs currently
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used to treat and prevent allergic diseases. However, these medications can show central side
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effects, and in certain cases do not result in efficient recovery.2
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Mast cells play a critical role as regulators of allergic diseases due to their wide
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distribution in various tissues.3 Allergic reactions result in mast cell degranulation, which
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leads to the rapid release of histamine and various inflammatory mediators such as
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leukotrienes, serotonin, chemokines, and proinflammatory cytokines, including interleukin
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(IL)-1β, IL-6, IL-8, and TNF-α. These mediators aggravate allergic responses through the
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infiltration, differentiation, and activation of immune cells.4 Therefore, modulation of mast
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cell degranulation and release of allergic mediators, is an effective treatment strategy for
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allergic diseases.
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Mitogen-activated protein kinases (MAPKs) signaling pathway is associated with several
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pivotal physiological processes such as proliferation, activation, degranulation, and migration.
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Extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 are
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major components of the MAPK signaling pathway.5-7 Extracellular stimuli activate MAPKs,
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which induce the degradation of IκB proteins and the nuclear translocation of nuclear factor-
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kappaB (NF-κB). Activation of NF-κB causes the transcription of target genes, including
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inflammatory mediators, which are crucial factors in the development of allergic responses.6, 3
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8, 9
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Arctium lappa (Arctii Fructus) seeds have been used as anti-inflammatory, diuretic, and
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detoxifying drugs in oriental medicine.10 The Arctii Fructus extract improves high
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fat/cholesterol diet-induced endothelial dysfunction and dermal extracellular matrix
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metabolism, which reduces wrinkles.11,12 Arctigenin (ARC) is one of the major active
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constituents present in the Arctii Fructus water (AFW) and ethanol extracts (AFE).13 ARC
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has anti-hypertension, anti-tumor, anti-osteoclastogenesis, and neuroprotective activities.14-19
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Specifically, fermented Arctii Fructus extract can suppress IgE-mediated allergic reaction in
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RBL‑ 2H3 cells.20 Moreover, ARC successfully inhibited ovalbumin (OVA)-induced
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anaphylaxis, compound 48/80-induced histamine release, and proinflammatory enzyme
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activities.21 However, the anti-allergic activities of Arctii Fructus and ARC on human mast
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cells and the underlying mechanisms are unclear. The aim of this study was to determine the
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effect of Arctii Fructus and ARC on mast cell-mediated allergic inflammation.
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MATERIALS AND METHODS
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Preparation of extracts and ARC isolation from Arctii Fructus. Dried and ground Arctii
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Fructus powder were obtained from Omniherb (Uiseong, Republic of Korea). AFW was
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prepared by extracting the water-soluble components of the Arctii Fructus powder in boiling
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water (100 g/L of water) at 100°C for 3 h. The solution was filtered using Whatman filter
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paper and lyophilized. To prepare the ethanol extract of Arctii Fructus (AFE), 100 g of Arctii
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Fructus was boiled in 70% ethanol (1 L) for 3 h and evaporated using a Rotary evaporator.
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The extract was freeze-dried at −56 °C and 9 mm Torr. ARC was isolated from A. lappa
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seeds as reported previously.21 ARC was identified by comparing the 1H-NMR spectrum
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data.23 AFW was dissolved in distilled water, whereas AFE and ARC were dissolved in 4
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DMSO (final concentration of DMSO is less than 0.1%).
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Reagents. Anti-phospho-ERK, phospho-JNK, phospho-p38, phosphor-IκBα, NF-κB, and
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Receptor interacting protein-2 (RIP2) antibodies were purchased from Cell Signaling
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Technology, Inc. (Danvers, MA, USA). Anti-histone, α-tubulin, ERK, JNK, p38, and caspase-
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1 antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Thiazolyl Blue
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Tetrazolium Bromide (MTT), PMA, A23187, DNP-IgE, DNP-BSA, and compound 48/80
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were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
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Cell culture. The human mast cell line (HMC-1) was provided by Eichi Morri (Osaka
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University, Japan), and the rat basophilic leukemia mast cell line RBL-2H3 was purchased
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from Korean Cell Line Bank (Seoul, Republic of Korea). These cell lines were cultured in
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10% FBS, 100 U/mL of penicillin and streptomycin in Iscove's Modified Dulbecco's Medium
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and Dulbecco's Modified Eagle's Medium, respectively. Cells were cultured in an incubator
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with 5% CO2/95% air at 37°C.
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Cell viability. Cells were seeded in 24-well microplates at a density of 5 × 104 cells/well and
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stabilized overnight. After 24 h of treatment with AFW, AFE, and ARC, cells were treated
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with MTT reagent (0.5 mg/mL) and incubated for 4 h. Formazan was lysed with DMSO, and
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colorimetric changes were measured using a VersaMax ELISA Microplate Reader at 540 nm.
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Histamine assay. Histamine production from mast cells was measured using the human
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histamine ELISA kit (ALPCO Diagnostics, NH, USA) as per the manufacturer’s protocol.
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HMC-1 cells were pretreated with AFW, AFE, and ARC for 1 h and stimulated with PMA 5
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plus A23187 (PMACI; PMA 50 nM and A23187 1 µM) for 24 h. RBL-2H3 cells were
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sensitized with DNP-IgE (100 ng/mL) for 12 h, and the samples were added to cells 1 h
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before DNP-BSA treatment. The conditioned medium was collected and used as a sample.
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Real-time RT-PCR. Total RNA was isolated from cells, and cDNA was synthesized using
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RNA-spinTM, Total RNA Extraction Kit, and Power cDNA Synthesis Kit (iNtRon Biotech,
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Seoul, Republic of Korea). Expression specific genes was determined by step-one plusTM
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real-time PCR systems (Applied Biosystems, Foster City, CA, USA). All data were
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normalized to GAPDH mRNA. The primer sequences are summarized in supporting
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information.
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Enzyme-linked immunosorbent assay (ELISA). Release of proinflammatory cytokines was
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measured by using a BD OptEIA ELISA kit (BD Pharmingen, San Diego, CA, USA)
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according to manufacturer’s protocol. Briefly, 96-well microplates were precoated with
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capture antibodies at 4°C overnight. After blocking with 5% FBS for 1 h, samples and
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standards were added and incubated for at least 3 h. The plates were washed with washing
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buffer and incubated with detection antibodies and streptavidin HRP for 1 h 30 min. This was
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followed by washing with 0.05% PBST five times, and the plates were incubated with TMB
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substrate reagent at 37°C for 30 min. Colorimetric development was blocked with a stop
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solution and measured using a microplate reader at 405 nm.
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Western blot analysis. Collected cells were washed and lysed using pro-prep protein
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extraction solution (iNtRon Biotech, Seoul, Republic of Korea) to extract total protein. Cell
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lysates were centrifuged at 13,000 rpm for 5 min, and the supernatants containing the proteins 6
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were obtained. Nuclear extracts were isolated using NE-PER Nuclear and Cytoplasmic
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Extraction Reagents (Pierce Thermo Scientific, Rockford, IL, USA). Total protein was
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quantified using BCA protein assay, mixed with a 5x sample buffer. Following SDS-
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polyacrylamide gel electrophoresis, proteins were transferred to PVDF membrane. Specific
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proteins were detected using primary antibodies and Horseradish Peroxidase secondary
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antibodies. Protein bands were photographed using the FluorChemTM M system
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(Proteinsimple, San Jose, CA, USA).
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Caspase-1 activity Assay. Caspase-1 colorimetric assay kit (Biovision, CA, USA) was used to
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determine caspase-1 activity according to the recommended instructions. Briefly, PMACI
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was added to AFW, AFE, and ARC-pretreated HMC-1 cells for 24 h, and harvested cells were
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lysed with lysis buffer. Caspase-1-specific peptide YVAD-pNA was used for the
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measurement of caspase-1 activity. Colorimetric changes were quantified using a microplate
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reader at 405 nm.
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Animals. ICR mice (five weeks, male) were purchased from Samtaco Korea (Osan, Republic
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of Korea). Mice were maintained in a laminar airflow room at 22 ± 1°C. An in vivo
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experiment was performed in accordance with the regulations issued by Wonkwang
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University guidelines (WKU14-100).
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Compound 48/80-induced anaphylactic shock. AFW, AFE (25–100 mg/kg), and ARC (10–
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50 mg/kg) were orally administered 1 h prior to injecting compound 48/80. Control mice
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received the same volume of 0.05% carboxymethyl cellulose. After intraperitoneal injection
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of compound 48/80 (8 mg/kg), mortality by anaphylactic shock was monitored for 1 h. 7
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Passive cutaneous anaphylaxis (PCA). A day prior to the experiment, hair was completely
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removed from the back of mice, and anti-dinitrophenyl (DNP)-IgE (0.5 µg) was injected into
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the dorsal skin. AFW, AFE (100 mg/kg), and ARC (50 mg/kg) were orally administered 1 h
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before the antigen challenge. DNP-BSA (1 µg) in saline containing 1% Evans blue was
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intravenously injected into each mouse via the tail vein. After killing, the dorsal skin was
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resected to evaluate the effect of the compounds on PCA. Blue dye in the dorsal skin was
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dissolved with formaldehyde for 24 h and colorimetric change was measured using a
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microplate reader at 620 nm. The inhibition rates of the compounds were calculated using the
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following equation: Inhibition rate (%) = 100 - (a/b × 100); ‘a’ is the average absorbance of
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the sample group, and ‘b’ is the average absorbance of the control group.
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Statistical analysis. Results are shown as the mean ± S.D. from at least three experiments. All
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statistical analyses were performed by using the Student’s t-test, and p < 0.05 indicated
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statistical significance.
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RESULTS
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Arctii Fructus extracts and ARC decrease histamine release in mast cells: Mast cell
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degranulation results in histamine release by antigen stimulation causing allergic reactions.4
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Therefore; we investigated whether AFW, AFE, and ARC can suppress the release of
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histamine from activated mast cells. MTT assay was performed to determine the effect of
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AFW, AFE, and ARC on the viability of HMC-1 cells. As shown in Figure 1A and B, AFW
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and AFE (2–50 µg/mL) did not result in any cytotoxicity after 24 h of incubation. Doses of
