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Cassia tora Seed Extract and Its Active Compound Aurantio-obtusin Inhibit Allergic Responses in IgE-Mediated Mast Cells and Anaphylactic Models Myungsuk Kim, Sue Ji Lim, Hee-Ju Lee, and Chu Won Nho* Natural Products Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangwon, Korea S Supporting Information *

ABSTRACT: Cassia tora seed is widely used due to its various biological properties including anticancer, antidiabetic, and antiinflammatory effects. However, there has been no report of the effects of C. tora seed extract (CTE) on immunoglobulin E (IgE)mediated allergic responses. In this research, we demonstrated the effects of CTE and its active compound aurantio-obtusin on IgE-sensitized allergic reactions in mast cells and passive cutaneous anaphylaxis (PCA). CTE and aurantio-obtusin suppressed degranulation, histamine production, and reactive oxygen species generation and inhibited the production and mRNA expression of tumor necrosis factor-α and interleukin-4. CTE and aurantio-obtusin also suppressed the prostaglandin E2 production and expression of cyclooxygenase 2. Furthermore, CTE and aurantio-obtusin suppressed IgE-mediated FcεRI signaling such as phosphorylation of Syk, protein kinase Cμ, phospholipase Cγ, and extracellular signal-regulated kinases. CTE and aurantioobtusin blocked mast cell-dependent PCA in IgE-mediated mice. These results suggest that CTE and aurantio-obtusin are a beneficial treatment for allergy-related diseases. KEYWORDS: aurantio-obtusin, Cassia tora, IgE-mediated allergic diseases, RBL-2H3 mast cells

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INTRODUCTION An allergy is an acquired hypersensitivity reaction of the immune system in response to harmless environmental substances.1 An allergy exhibits in various forms ranging from allergic rhinitis and conjunctivitis, urticaria, and asthma to severe anaphylaxis,1,2 causing an enormous socioeconomic burden. Markedly, mast cells play a crucial role in causing immunoglobulin E (IgE)-mediated allergic responses.3 Mast cell activation can be induced by cross-linking of allergen-IgE bound by high-affinity IgE receptors (FcεRI) on the cells.4 Activation of mast cells initiates a cascade of intracellular signalings, including phosphorylation of tyrosine kinases such as Lyn and Syk, the phospholipase Cγ (PLCγ), protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs).4 Once activated, mast cells generate several biologically active products, namely, cytoplasmic granule-derived mediators, lipidderived mediators, and proinflammatory cytokines.5 Cytoplasmic granules comprise histamine, which is a key marker of allergic responses and can trigger increase of vascular permeability. Inflammatory lipid-derived mediators, such as leukotrienes (LTs) and prostaglandins (PGs), stimulate erythema and vasodilation.1 Stimulated mast cells also generate different types of proinflammatory chemokines and cytokines, which have the capability to gather other immune cells either directly or indirectly. Therefore, mast cells lead to acute inflammation and chronic inflammation.5 The rat basophilic leukemia mast cell line (RBL-2H3) is normally used as an in vitro model of IgE-mediated allergic responses.6 RBL-2H3 cells present high-affinity FcεRI receptors for IgE on the cell surface, and cross-linking of the IgE-FcεRI complex induced by antigens initiates several cascades of intracellular events that lead to mast cell degranulation and © XXXX American Chemical Society

consequently the secretion of proinflammatory mediators of allergic responses.7 Cassia tora seed is widely used to relax the bowels, reduce inflammation in the liver, and improve eyesight, and is drunk as a roasted tea in Korea and China. It has been used as a laxative and a tonic, and is also a popular health tea drink.8 C. tora seed also has an anti-hyperlipidemic effect related to decreased levels of serum low-density lipoprotein cholesterol and triglycerides in diabetic patients.9,10 C. tora seed has a neuroprotective effect in mice with transient cerebral global ischemia based on its antiinflammatory action.11 C. tora seed extract (CTE) and its isolated constituents have antimicrobial,12 antioxidant,13 hepatoprotective,14 and hypolipidemic15 properties. C. tora seed contains anthraquinone compounds, such as emodin, chrysophanol, aurantio-obtusin, chryso-obtusin, obtusin, and obtusifolin.16 However, the effects of C. tora seed and its components on mast cell activation have not been studied. In this study, we assessed the antiallergic properties of CTE and aurantio-obtusin by investigating their effects on IgE-stimulated RBL-2H3 cells in vitro.

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MATERIALS AND METHODS

Reagents and Materials. Reagents were obtained as follows: antidinitrophenyl (DNP)-specific immunoglobulin E (IgE), DNPbovine serum albumin (DNP-BSA), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), 1,4-piperazinediethanesulfonic acid (PIPES), and 4amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine (PP2, all from Sigma-Aldrich, St. Louis, MO, USA), and fetal bovine Received: August 5, 2015 Revised: October 2, 2015 Accepted: October 3, 2015

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DOI: 10.1021/acs.jafc.5b03836 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. (A) Chemical structure of anthraquinones and (B) HPLC chromatogram from C. tora seed extract (CTE). serum (FBS; Hyclone, South Logan, UT, USA), penicillin (Invitrogen, Carlsbad, CA, USA), Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan, UT, USA), and phospho-Syk (Tyr525/526) antibody, phospho-phospholipase C (PLC)γ (Tyr783) antibody, phospho-PKD/PKCμ (Ser916) antibody, phosphor-SAPK/JNK (Thr183/Tyr185) antibody, SAPK/JNK (Thr183/Tyr185) antibody, phospho-p44/42 MAPK antibody (Erk1/2), p44/42 MAPK (Erk1/2) antibody, phospho-p38 antibody, p38 antibody, and β-actin antibody (all from Cell Signaling Technology Inc., Danvers, MA, USA). Plant Material. Seeds of C. tora, collected in Yeongcheon, Gyeongsang Province, Korea, were purchased from a commercial supplier in Gyeongdong market, Seoul, Korea, in August 2013 and were identified by one of the authors (H.-J.L.). A voucher specimen was deposited at the Korea Institute of Science and Technology (KIST), Gangneung Institute. Extraction was performed by sonication using an RK158S ultrasonic bath (Bandelin, Germany). Extraction and Isolation of Anthraquinones from C. tora Seeds. One kilogram of the dried seeds of C. tora was extracted with 1 L of 95% ethanol at room temperature and filtered through Whatman No. 1 filter paper (GE Healthcare Life Sciences, PA, USA). The filtrate

was concentrated under reduced pessure by rotary evaporation at 40 °C, and 95 g of EtOH extract (CTE) was obtained. Isolation of anthraquinones (Figure 1) from C. tora was done as previously described.16 Cell Culture. RBL-2H3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) via the Korean Cell Line Bank (KCLB No. 22256, Seoul, Korea). Cells were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin. Cells were grown in 75 cm2 culture flasks at 37 °C with 5% CO2 in a humidified atmosphere. Cell Viability Assay. Cell viability was checked using a 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St Louis, MO, USA). RBL-2H3 cells were grown in 24-well plates (2 × 105 cells/mL) for 24 h. After treatment of the cells with various concentrations of CTE and aurantio-obtusin for 6 h, the cells were washed and then treated with 200 μL of MTT (0.5 mg/mL) and cells were incubated for an additional 4 h. Cells were then washed, and the insoluble formazan products were dissolved in 200 μL of DMSO. Absorbance was measured by spectrophotometry at B


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Figure 2. Effects of CTE and aurantio-obtusin on β-hexosaminidase and histamine release. (A) The degranulation was determined by measuring βhexosaminidase release in DNP-IgE-sensitized RBL-2H3 cells. The RBL-2H3 cells were treated in the presence or absence of CTE (5, 10, or 20 μg/ mL) for 1 h and then stimulated for 1 h with DNP-BSA. (B) The RBL-2H3 cells were treated in the presence or absence of gluco-aurantioobtusin (1), gluco-obtusifolin (2), aurantio-obtusin (3), chryso-obtusin (4), obtusin (5), or obtusifolin (6) (10, 20, or 40 μM) for 1 h and then stimulated for 1 h with antigen. (C, D) Histamine release was determined with enzyme-linked immunosorbent assay kit. The RBL-2H3 cells were treated in the presence or absence of CTE (5, 10, or 20 μg/mL) and aurantio-obtusin (5, 10, or 20 μM) for 1 h and then stimulated for 1 h with antigen. PP2 (10 μM) is a general Src-family kinase inhibitor. Results are expressed as the mean ± SD of three independent experiments (##p < 0.01, *p < 0.05, and **p < 0.01). Measurement of Cytokine Release. IgE-sensitized RBL-2H3 cells were incubated with CTE and aurantio-obtusin (various concentrations) for 1 h and stimulated with DNP-BSA for 4 h. Interleukin (IL)-4 and tumor necrosis factor (TNF)-α concentrations in cell culture supernatants were measured using enzyme-linked immunosorbent assay kits (Abcam, U.K.). Measurement of Reactive Oxygen Species (ROS) Production. RBL-2H3 cells were pretreated with CTE and aurantio-obtusin for 1 h before being sensitized overnight with 0.2 μg/mL of DNPspecific IgE antibody. The cells were washed with PBS, and stained with 40 μM DCFH-DA for 30 min. For fluorescence staining assay, cells were then stimulated with 1 μg/mL of DNP-BSA for 30 min, and changes in fluorescence intensity were measured using a microplate reader (Bio-Tek Instruments, Winooski, VT) at excitation and emission wavelengths of 485 and 530 nm, respectively. Reverse Transcription-Polymerase Chain Reaction. (RT-PCR). RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and quantified by spectrophotometry at 260 nm. The cDNA was synthesized in a 20 μL reaction containing 1 μg of total RNA, oligo (dT), and reverse transcriptase premix (ElpisBiotech., Inc., Taejeon, Korea). Reverse transcription (RT) was initiated at 70 °C for 5 min, followed by incubation at 42 °C for 60 min, and was terminated at 94 °C for 5 min. Amplification of the cDNA products (5 μL) by PCR was performed with a PCR premix

550 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT). Measurement of Histamine and β-Hexosaminidase Release. RBL-2H3 cells were sensitized with 0.2 μg/mL monoclonal DNP-IgE. Cells were washed with modified PIPES buffer (25 mM PIPES, pH 7.2, 119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2·6H2O, 1 mM CaCl2, 5.6 mM glucose, 40 mM NaOH, and 0.1% BSA). Cells were treated with CTE or compounds 1−6 at the indicated concentrations for 1 h at 37 °C. After incubation, cells were challenged with 0.2 μg/mL DNP-BSA for 1 h at 37 °C. Histamine was then detected using an enzyme immunoassay kit (Oxford Biomedical Research, Rochester Hills, MI). Histamine release was expressed as a percentage of the total histamine produced by unstimulated cells. To determine βhexosaminidase release, supernatants and lysed pellets were aliquoted into 96-well plates. Samples were mixed with substrate solution (1 mM p-nitrophenyl N-acetyl-β-D-glucosamine in 0.05 M citrate buffer, pH 4.5) and incubated for 1 h at 37 °C. Reactions were terminated by the addition of 0.05 M sodium carbonate buffer, pH 10. Absorbance was measured by spectrophotometry at 405 nm. Measurement of Prostaglandin E2. Cell culture medium was collected, and the secretion of prostaglandin E2 (PGE2) was determined using competitive radioimmunoassay kits (R&D Systems, MN, USA, and Cayman Chemical, MI, USA, respectively) according to the manufacturers’ protocol. C


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Figure 3. Effects of CTE and aurantio-obtusin on PGE2 secretion and COX-2 mRNA expression. RBL-2H3 cells were treated with CTE (5, 10, or 20 μg/mL) and aurantio-obtusin (5, 10, or 20 μM) for 1 h and then stimulated for 4 h with antigen. (A, B) PGE2 secretion was determined by enzymelinked immunosorbent assay kit. (C, D) COX-2 mRNA level was determined with RT-PCR. PP2 (10 μM) is a general Src-family kinase inhibitor. Results are expressed as the mean ± SD of three independent experiments (##p < 0.01, *p < 0.05, and **p < 0.01). Laboratories, Hercules, CA, USA). Equal amounts of protein (30 μg) were loaded in each sample, separated by 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). Blotted membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, and then incubated with primary antibodies for 16 h at 4 °C. After three washes in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. Proteins were detected with enhanced chemoluminescence (Amersham Biosciences, Little Chalfont, U.K.) and visualized using an ECL Advanced system (GE Healthcare, Hatfield, U.K.). Statistical Analysis. Each experiment was performed at least three times. Results are expressed as the mean ± standard deviation (SD). Vehicle (DMSO) and treated groups were compared by one-way analysis of variance followed by Scheffe’s test (SPSS 17.0, Chicago, IL, USA). ##p < 0.01, *p < 0.05, and **p < 0.01 were considered statistically significant. ##p < 0.01 compared with the control group (DNP-IgE treatment alone); *p < 0.05 and **p < 0.01 compared with the DNP-IgE and DNP-BSA stimulated group.

(Elpis-Biotech) and the following primer pairs: TNF-α forward, 5′CAA GGA GCA GAA GTT CCC AA-3′, and TNF-α reverse, 5′-CGG ACT CCG TGA TGT CTA AG-3′ (500 bp); IL-4 forward, 5′-ACC TTG CTG TCA CCC TGT TC-3′, and IL-4 reverse, 5′-TTG TGA GCG TGG ACT CAT TC-3′ (351 bp); COX-2 forward, 5′- TGA CTG TAC CCG GAC TGG AT-3′, and COX-2 reverse, 5′-CAT GGG AGT TGG GCA GTC AT-3′ (322 bp); β-actin forward, 5′AGC CAT GTA CGT AGC CAT CC-3′, and β-actin reverse, 5′-TCT CAG CTG TGG TGG TGA AG-3′ (227 bp). Before PCR amplification, primers for cytokines and β-actin were denatured at 94 °C for 5 min. Amplification consisted of 28 cycles of denaturation at 94 °C for 30 s, annealing for 1 min, and extension at 72 °C for 1 min, followed by a final 5 min extension at 72 °C. PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). PCR products were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination. Densitometry was performed using DIG chemiluminescent film (volume of all markers/volume of β-actin). Passive Cutaneous Anaphylaxis (PCA) in Mice. DNP-IgE (0.5 mg) was subcutaneously injected onto the ears of BALB/c mice (4week-old male). After 24 h, the mice were orally administered with CTE (200 mg/kg), aurantio-obtusin (50 mg/kg), or cetirizine (20 mg/kg). After 1 h, the mice were injected intravenously with DNPBSA (250 μg) in PBS containing Evans Blue (5 mg/mL). After 60 min, the ear tissue was removed and absorbed dye was extracted overnight in formamide (700 μL) at 63 °C. The absorbance was measured at 620 nm. Animal experimental procedures were approved by the Animal Care and Use Committee of KIST. Western Blot Analysis. RBL-2H3 cells were lysed in RIPA buffer (containing 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing protease inhibitors and then placed on ice for 10 min. Lysate protein concentrations were determined by Bradford protein assay (Bio-Rad

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RESULTS Identification of Anthraquinones in C. tora Seeds. Six anthraquinones were identified in C. tora using liquid chromatography−mass spectrometry, namely, gluco-aurantioobtusin, gluco-obtusifolin, aurantio-obtusin, chryso-obtusin, obtusin, and obtusifolin (Figure 1). Effects of CTE and Aurantio-obtusin on Degranulation and Histamine Release in IgE-Sensitized RBL-2H3 Cells. The effects of CTE and aurantio-obtusin on cell cytotoxicity were assessed by the colorimetric MTT assay. D


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Figure 4. Effects of CTE and aurantio-obtusin on TNF-α and IL-4 production and mRNA expression. RBL-2H3 cells were treated with CTE and aurantio-obtusin for 1 h and then stimulated with DNP-BSA for 4 h. (A−D) TNF-α and IL-4 production was determined by enzyme-linked immunosorbent assay kit. (E, F) mRNA levels of TNF-α and IL-4 were determined with RT-PCR. PP2 (10 μM) is a general Src-family kinase inhibitor. Results are expressed as the mean ± SD of three independent experiments (##p < 0.01, *p < 0.05, and **p < 0.01).

CTE (up to 20 μg/mL) or aurantio-obtusin (up to 20 μM) had no effect on RBL-2H3 cell viability (Figures S1 and S2). To verify the effects of CTE and its isolated compounds on degranulation of mast cell, we examined their potential to suppress the β-hexosaminidase release. CTE suppressed βhexosaminidase release in IgE-sensitized mast cells in a dosedependent manner (Figure 2A). In addition, aurantio-obtusin inhibited β-hexosaminidase release more than gluco-aurantioobtusin, gluco-obtusifolin, chryso-obtusin, obtusin, and obtusifolin in IgE-sensitized mast cells (Figure 2B). Thus, additional experiments were carried out using aurantio-obtusin.

The effects of CTE and aurantio-obtusin on histamine release were also assessed to investigate potential antiallergic activities. CTE and aurantio-obtusin repressed histamine release in IgEsensitized mast cells (Figures 2C and 2D). Inhibitory Effects of CTE and Aurantio-obtusin on PGE 2 Production and Cyclooxygenase 2 (COX-2) Expression in IgE-Sensitized RBL-2H3 Cells. In antigenstimulated mast cells, the arachidonate cascade is initiated via the FcεRI receptor and recruits inflammatory cells, such as neutrophils, Th2 cells, eosinophils, and basophils.17 Therefore, these arachidonic acid metabolites contribute to inflammation E


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Journal of Agricultural and Food Chemistry in allergic diseases, and suppression of their production is essential to prevent or treat allergic diseases. To investigate the effects of CTE and aurantio-obtusin on the production of PGE2, RBL-2H3 cells were sensitized with 200 ng/mL DNPIgE overnight and treated with CTE and aurantio-obtusin for 1 h followed by the treatment with DNP-BSA for 24 h. CTE and aurantio-obtusin significantly inhibited PGE2 production in a dose-dependent manner in IgE-stimulated RBL-2H3 cells (Figures 3A and 3B). The effects of CTE and aurantio-obtusin on COX-2 expression were confirmed by RT-PCR. DNP-BSA increased COX-2 mRNA expression in IgE-sensitized mast cells. However, treatment with CTE and aurantio-obtusin reduced IgE-mediated upregulation of COX-2 expression (Figures 3C and 3D). These results indicated that CTE and aurantio-obtusin inhibit the release of pro-inflammatory lipid mediators from activated mast cells and suggest that they may have an antiallergic effect. Inhibitory Effects of CTE and Aurantio-obtusin on the Production and mRNA Expression of Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-4 in IgE-Sensitized RBL-2H3 Cells. Allergic inflammatory responses are induced by proinflammatory cytokines from activated mast cells. TNF-α and IL-4, which are proinflammatory cytokines, are essential in allergy-related diseases.18 We therefore determined whether CTE and aurantio-obtusin inhibited the production and mRNA expression of TNF-α and IL-4 in IgE-stimulated RBL-2H3 cells. Consistently with the previous report,19 the increase of the production and expression of TNF-α and IL-4 was obvious by DNP-BSA stimulation in mast cells. CTE and aurantio-obtusin decreased both the production (Figures 4A−D) and mRNA expression (Figures 4E and 4F) of TNF-α and IL-4. These results showed that CTE and aurantio-obtusin have an inhibitory effect on the production and expression of proinflammatory cytokines in mast cells. Effects of CTE and Aurantio-obtusin on Reactive Oxygen Species (ROS) Production in IgE-Sensitized RBL2H3 Cells. Several reports suggested that activated mast cells have increased intracellular ROS levels.20 Moreover, ROS augments histamine release in mast cells,21 and H2O2 can regulate phosphorylation of PLCγ.22 We next examined to investigate whether CTE and aurantio-obtusin attenuate ROS production following IgE-mediated mast cell activation using the DCFH-DA fluorescent indicator. Fluorometer analysis showed that the level of ROS increased from 10.60% in nonstimulated cells to 58.62% in antigen-sensitized cells. However, ROS production was significantly reduced by CTE or aurantio-obtusin treatment in a dose-dependent manner (Figures 5A and 5B). Effects of CTE and Aurantio-obtusin on FcεRI Signaling in IgE-Sensitized RBL-2H3 Cells. Aggregation of FcεRI consecutively initiates IgE-mediated phosphorylation of tyrosine kinases such as Syk and Lyn, and subsequently tyrosine phosphorylation of cellular proteins such as PLCγ and PKC.19 PP2, a general Scr-family kinase inhibitor, significantly suppresses Syk phosphorylation.19 To determine the mechanisms underlying CTE- and aurantio-obtusin-mediated inhibition of allergic responses, we examined several intracellular signals. Phosphorylation of Syk, PKD/PKCμ, and PLCγ was induced in antigen-stimulated RBL-2H3 cells, and this phosphorylation was inhibited by treatment with CTE and aurantio-obtusin (Figures 6A and 6B). We also examined the effects of CTE and aurantio-obtusin on MAPKs because of their role in the production of TNF-α and IL-4.23 Among

Figure 5. Effect of CTE and aurantio-obtusin on intracellular ROS production. (A, B) RBL-2H3 cells were treated with CTE and aurantio-obtusin for 1 h and then stained with 40 μM 2′7′dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) for 30 min. The stained cells were then stimulated with DNP-BSA for 10 min. After 30 min, ROS levels were analyzed by flow cytometry. PP2 (10 μM) is a general Src-family kinase inhibitor. Results are expressed as the mean ± SD of three independent experiments (##p < 0.01, *p < 0.05, and **p < 0.01).

phosphorylation of ERK, JNK, and p38 induced by antigen stimulation, phosphorylation of ERK was only suppressed by treatment with CTE and aurantio-obtusin (Figures 6C and 6D). These results suggest that CTE and aurantio-obtusin suppresses mast cell activation by inactivation of FcεRI signaling and ERK in mast cells. Effect of CTE and Aurantio-obtusin on IgE-Mediated PCA in Mice. PCA is generally used to measure local allergic responses in vivo.24 To examine whether CTE and aurantioobtusin repressed allergic reactions, we introduced a IgEmediated PCA model. IgE was subcutaneously injected onto the left ear tissue of mouse, followed by injection of DNP-BSA containing 5% Evans Blue. CTE and aurantio-obtusin suppressed the IgE-sensitized PCA response (Figures 7A and 7B). Cetrazine, a typical antihistamine drug, was used as a positive control.25

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DISCUSSION Type I hypersensitivity is triggered by inflammatory chemical mediators (cytokines, ILs, LTs, and PGs) secreted from immune cells such as eosinophils, basophils, and mast cells. F


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Figure 6. Effect of CTE and aurantio-obtusin on FcεRI Signaling. (A, B) RBL-2H3 cells were treated with CTE and aurantio-obtusin for 1 h and then stimulated with DNP-BSA for 10 min. Phosphorylation of Syk, PLCγ, and PKD/PKCμ was determined with Western blot analysis. (C, D) RBL-2H3 cells were treated with CTE and aurantio-obtusin for 1 h and then stimulated with DNP-BSA for 30 min. Phosphorylation of JNK, ERK, and p38 was determined with Western blot analysis. PP2 (10 μM) is a general Src-family kinase inhibitor.

histamine.43 CTE and aurantio-obtusin suppressed the secretion of β-hexosaminidase (Figures 2A and 2B) and histamine (Figures 2C and 2D). These results suggest that the antiallergic activities of CTE and aurantio-obtusin are caused by the inhibition of histamine release, which causes smooth muscle contraction, vascular permeability, and inflammation. The FcεRI receptor signals through Syk, Lyn, and PLCγ to regulate mast cell activation.17 The FcεRI receptor aggregates to IgE and starts intracellular signal cascade to derive allergic reactions. Stimulated FcεRI initiates phosphorylation of Syk, which binds to immunoreceptor tyrosine-based activation motifs present on the β and γ chains of FcεRI. Consecutively, Syk derives phosphorylation of PKC and PLCγ.44 Remarkably, CTE and aurantio-obtusin declined the phosphorylation of Syk, PLCγ, and PKD/PKCμ (Figures 6A and 6B). These results suggest that CTE and aurantio-obtusin have antiallergic effects in IgE-sensitized mast cells by inhibiting the phosphorylation of Syk, PKC, and PLCγ, and consequently inhibiting mast cell degranulation. CTE and aurantio-obtusin also decreased production of the inflammatory mediator PGE2, which is an arachidonic acid metabolite, via suppression of the COX pathways (Figures 3A and 3B).45 Furthermore, CTE and aurantio-obtusin inhibited mRNA COX-2 expression, a key enzyme in these pathways (Figures 3C and 3D). These results indicate that CTE and aurantio-obtusin inhibit generation of inflammatory lipid mediators by inhibiting the COX pathways.

Particularly, mast cells are key players in the control of allergic related reactions.26 A recent study revealed that mast cellreleased chemical mediators (histamines, cytokines, and proteases) play a crucial role during allergic mediated inflammation in mast cell-deficient mice.27 Therefore, several studies have reported specific mediators of mast cell activation and identified novel compounds that inhibit these allergic mediators.3,28,29 Anthraquinones are predominantly reported in and isolated from the seeds of Cassia tora L. (syn. Senna tora) and Cassia obtusifolia L. (syn. Senna obtusifolia),30,31 and may possess antiinflammation, antioxidant, and anticancer properties.32−34 Moreover, anthraquinones inhibit chemical mediators of mast cell activation. For instance, emodin, rhein, and citreorosein, three naturally occurring anthraquinone derivatives, suppress the IgE-mediated anaphylactic reaction, mast cell activation, and LT generation.35−37 Among anthraquinone derivatives isolated from C. tora, aurantio-obtusin is the major compound. It is also used as a quality control marker of C. tora and reportedly has antihypertensive, hepatoprotective, antimutagenic, osteogenic, and estrogenic activities.38−42 However, the effects of C. tora seed and aurantio-obtusin on allergic diseases have not been studied. In this study, we assessed the inhibitory effects of CTE and its active compound aurantio-obtusin on allergic inflammation in mast cells. Activation of FcεRI receptors by the high-affinity binding and aggregation of IgE triggers a signaling cascade that led to degranulation and production of inflammatory mediators, such as inflammatory cytokines, lipid-derived mediators, and G


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Figure 7. Effect of CTE and aurantio-obtusin on IgE-mediated passive cutaneous anaphylaxis (PCA). (A) Mice were sensitized with IgE for 1 h and then orally administered with CTE (200 mg/kg), aurantio-obtusin (50 mg/kg), or cetrizine (20 mg/kg) for 1 h. The ears of mice absorbed with Evans Blue were photographed. (B) The ear absorbed dye was extracted with formamide. The quantification data is presented as % of IgE- sensitized control. Cetrizine (20 mg/kg) is an antihistamine reference drug. Data are expressed as the mean ± SD (n ≥ 6) (*p < 0.05 and **p < 0.01).

and aurantio-obtusin suppressed IgE-mediated phosphorylation of ERK, indicating that these compounds mitigate mast cell activation by inhibiting MAPK pathway (Figures 6C and 6D). In conclusion, CTE and its active compound aurantioobtusin suppressed degranulation, the generation of histamine, inflammatory lipid mediators, and proinflammatory cytokines, and intracellular ROS production in IgE-sensitized mast cells. CTE and aurantio-obtusin also decreased signaling cascade from the FcεRI receptor by inhibiting the activation of Syk, PLCγ, PKC, and MAPK, all of which intermediate allergic reactions in IgE-sensitized mast cells. Our results suggest that CTE and aurantio-obtusin are beneficial therapeutic agents to prevent or treat IgE-sensitized allergic disorders.

CTE and aurantio-obtusin inhibited both the production and mRNA expression of the proinflammatory mediators TNF-α and IL-4 (Figure 4). TNF-α and IL-4 play an essential role in activating and continuing allergic responses in mast cells. IL-4 derives T-cell development and switches B-cells to the IgE isotype. TNF-α also induces physiological immune reactions by stimulating tissue fibrosis and leukocyte infiltration, and initiates cytokine-mediated inflammatory states by promoting cytokine production.46 Even though inflammation is a physiological defense system against pathogens, overexpression of cytokines can also cause serious inflammatory disorders such as asthma and atopic dermatitis. Our results indicate that these compounds are beneficial for the treatment of allergic disorders. Furthermore, the function of ROS as an inducer of degranulation and Ca2+ increase was revealed in recent studies.47,48 In the meantime, prevention of ROS production reportedly suppresses IgE-mediated histamine secretion.25 Thus, blockade of ROS production is regarded to be significant for inhibition of mast cell activation. CTE and aurantio-obtusin effectively reduced ROS production in IgE-sensitized RBL-2H3 cells. Several studies reported that CTE and aurantio-obtusin have an antioxidant property,49,50 which may result from their high ROS-elimination activity. These results suggest that the antioxidant property of CTE and aurantio-obtusin results in the inhibition of mast cell activation. MAPK signaling is a key therapeutic target in allergic diseases.51 Consequently, to examine the mechanism by which CTE and aurantio-obtusin suppress mast cell activation, phosphorylation of ERK, JNK, and p38 was assessed. CTE

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03836. Cell viability of CTE and aurantio-obtusin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Natural Products Research Center, Korea Institute of Science and Technology Gangneung Institute, 679 Saimdang-ro, Gangneung, Gangwon-do, Republic of Korea. Phone: 82-33650-3651. Fax: 82-33-650-3679. E-mail: [email protected]. H


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This work was supported by an intramural grant (2Z04381) from Korea Institute of Science and Technology, Gangneung Institute. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DMSO, dimethyl sulfoxide; FcεRI, Fc receptor for IgE; IL, interleukin; MAPK, mitogen-activated protein kinases; RBL, rat basophilic leukemia; TNF, tumor necrosis factor; IgE, immunoglobulin E; PLC, phospholipase; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; JNK, c-Jun Nterminal kinase; PGs, prostaglandins; COX-2, cyclooxygenase 2

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