TY - JOUR
AU - Arai, Hirofumi
AB - ABSTRACT Histamine and leukotrienes (LTs), the chemical mediators released from mast cells, play an important role in type-I allergies such as hay fever. Echinacea purpurea (EP) has traditionally been used for herbal tea and has been reported to show biological functions. We evaluated the inhibitory activity of water extracts of EP petals, leaves, and stems against the chemical mediators released from mast cell lines. Petal and leaf extracts exhibited a significant inhibitory effect on histamine release from the stimulated cells, while the stem extract did not exert any effect. Activity of the petal extract was much stronger than that of the leaf extract. All the extracts significantly suppressed LTB4 production in the stimulated cells and displayed similar activities. The petal extract decreased Syk phosphorylation and Ca2+ influx associated with signal transduction in the stimulated cells. These results suggest that EP petal extract may have a relieving effect on allergic symptoms. Graphical Abstract Open in new tabDownload slide Petal extract of Echinacea purpurea significantly inhibited chemical mediators released from stimulated mast cells, in which the suppression of tyrosine phosphorylation and Ca2+ influx were associated. Graphical Abstract Open in new tabDownload slide Petal extract of Echinacea purpurea significantly inhibited chemical mediators released from stimulated mast cells, in which the suppression of tyrosine phosphorylation and Ca2+ influx were associated. allergy, Echinacea purpurea, histamine, leukotriene, mast cell Abbreviations Abbreviations AA: arachidonic acid DNP: 2,4-dinitrophenyl DPPH: 2,2-diphenyl-1-picrylhydrazyl EDTA: ethylenediaminetetraacetic acid EP: Echinacea purpurea FcεRI: Fcε receptor I FBS: fetal bovine serum GAE: gallic acid equivalent IgE: immunoglobulin E LOX: lipoxygenase LT: leukotriene OPA: o-phthalaldehyde PBS: phosphate-buffered saline SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis Syk: spleen tyrosine kinase TBS: Tris buffered saline Allergy is a disorder of immune response that is classified into several types according to the mechanism involved (Gell and Coombs 1968; Uzzaman and Cho 2012; Dispenza 2019). Immediate hypersensitivities such as hay fever and food allergies are categorized as type-I allergies, in which mast cells and basophilic leukocytes play an important role in the allergic reaction (Gonzalez-de-Olano and Alvarez-Twose 2018). The specific binding of antigens, such as pollens, to immunoglobulin E (IgE) antibodies bound to Fcε receptor I (FcεRI) on the cell membrane induces cell stimulation by cross-linking of IgEs. This triggers the intracellular signal transduction, such as the phosphorylation of proteins, followed by Ca2+ influx into the cytoplasm. The increase in Ca2+ concentration causes the release of stored histamines from the granules through degranulation (Siraganian 2003). The influx of Ca2+ also induces the release of arachidonic acid (AA) from phospholipids of the cell membrane by activating phospholipase A2. AAs are oxidized by 5-lipoxygenase (LOX), and leukotrienes (LTs) such as LTB4 are then produced through cascade reactions (Werz 2002), which are secreted into the extracellular space. Histamine and LTs act as chemical mediators in type-I allergies, causing bronchoconstriction, dilatation, and hyperpermeability of blood vessels, leukocyte chemotaxis, and extension of inflammation involved in various allergic symptoms such as hypersecretion of mucus, sneezing, and cough (Galli, Tsai and Piliponsky 2008; Amin 2012; Galli and Tsai 2012). Functional foods have received particular attention in recent years due to their potential in relieving allergic symptoms instead of using symptomatic drug therapy because foods may have fewer side effects. Echinacea is a perennial flowering plant of the Asteraceae family native to North America and is commonly called purple coneflower. Native Americans have used Echinacea as a traditional medicine for various diseases such as colds (Kindscher 1989; Borchers et al. 2000; Kligler 2003). Today, Echinacea is widely cultivated all over the world and has been utilized for herbal tea that is made of hot water extracts of the whole powdered plant including petals, leaves, stems, and roots, and the extracts have been used to improve the respiratory and immune systems (Barnes et al. 2005; Sharifi-Rad et al. 2018). It has been suggested that the extracts of Echinacea purpurea (EP), a major species of Echinacea, exert various biological functions such as antibacterial, antioxidant, and anti-inflammatory activities (Woelkart and Bauer 2007; Sharma et al. 2009; Todd et al. 2015). The substances responsible for these functions are polyphenols such as caffeic acid derivatives including chicoric acid and hydrophobic alkylamides including isobutylamides which are abundant in the roots (Lee et al. 2015; Liu et al. 2017; Olah et al. 2017). Recently, (Gulledge et al. (2018) have reported that EP root extract and its alkylamide can suppress mast cell degranulation in vitro. However, the effect of the aerial part extracts of EP on type-I allergies is unclear. In the present study, we investigated the inhibitory activity of water extracts prepared from EP petals, leaves, and stems against the release of histamine and LTB4 from mast cells in vitro. Materials and methods Materials Folin–Ciocalteu's reagent was purchased from Nacalai Tesque (Kyoto, Japan). Chicoric acid was obtained from Tokyo Chemical Industry (Tokyo, Japan). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was supplied by Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were of reagent grade. Preparation of Echinacea purpurea extracts EP cultivated in Kumamoto, Japan, was obtained from a market. Extracts were individually prepared from the petals, leaves, and stems of EP as follows. Fifty grams of chopped frozen petals, leaves, and stems were mixed with 500 mL of distilled water at 80°C for 2 h. The extracts were then sonicated for 5 min and filtered (Filter paper No. 1, 125 mm, Advantec, Tokyo, Japan). Extraction was carried out with the residues by the same procedure. Extraction liquids were combined and freeze-dried. The yields of EP petals, leaves, and stems, were 5.58, 6.65, and 12.24%, respectively. Histamine release assay Inhibitory activity of the EP extracts against the release of histamine was evaluated according to the method described by Byeon et al. (2009) and Matsuo et al. (1997). The rat basophilic leukemia cell line RBL-2H3 was obtained from the JCRB Cell Bank (Tokyo, Japan). The cells were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Logan, UT, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin and maintained at 37°C under 5% CO2 and 95% air. To sensitize RBL-2H3 cells with IgE, cells suspended in the medium containing a mouse anti-2,4-dinitrophenyl (DNP) IgE monoclonal antibody (Yamasa, Choshi, Japan) were seeded in a 24-well cell culture plate at a density of 4 × 105 cells/well, and cultured for 20 h. The cells in each well were washed twice with phosphate-buffered saline (PBS, pH 7.4). EP extracts dissolved in Tyrode buffer (450 µL) consisting of 137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 12 mM NaHCO3, 1 mM MgCl2, 1.8 mM CaCl2, 5.6 mM glucose, and 0.05% bovine serum albumin (BSA), pH 7.2, was added to each well. After incubation at 37°C for 10 min, 50 µL of DNP-BSA (Calbiochem, Darmstadt, Germany) in Tyrode buffer (2 µg/mL) was added, and the cells were stimulated for 20 min at 37°C. The reaction was terminated by cooling the plate on ice. Tyrode buffer (50 µL) without DNP-BSA was used for the measurement of spontaneous histamine release from the cells without stimulation. For the determination of the total amount of histamine stored in the granules, 50 µL of 5% Triton X-100 was added to each well instead of Tyrode buffer, and the cells were incubated for 20 min on ice and lysed by pipetting. The supernatants after stimulation or the cell lysate were transferred to microtubes and centrifuged at 300 × g at 4°C for 5 min or at 20 800 × g at 4°C for 10 min, respectively. The histamine contents in the supernatants were then subjected to HPLC analysis as follows. The sample solution (100 µL) was mixed with 50 µL of 8 µM 1-methylhistamine as an internal standard and 50 µL of 200 mM N-acetyl-l-cysteine. The histamine in the solution was measured by reversed-phase HPLC with fluorescence detection (von Vietinghoff, Gabel and Aschenbach 2006). HPLC was performed on a polymer-based column (Shodex ODP-50-4E, 4.6 × 250 mm) maintained at 50°C. Five microliters of sample solution was injected and eluted with methanol/water (35/65, v/v) containing 30 mM Na2B4O7 and 0.2 mM o-phthalaldehyde (OPA) at a flow rate of 0.7 mL/min. The excitation and emission wavelengths for detection (RF-10AXL, Shimadzu, Kyoto, Japan) were 340 and 450 nm, respectively. LTB4 production assay Inhibitory activities of the EP extracts against LTB4 production were evaluated according to a previously described method with some modifications (Takasugi et al. 2018). The mouse mast cell line PB-3c obtained from the JCRB Cell Bank was cultured in RPMI-1640 with 2 mM L-glutamine and 25 mM HEPES (Wako) containing 10% FBS (HyClone), 1% MEM nonessential amino acids (Gibco, NY, USA), 1 mM sodium pyruvate (Gibco), 0.0035 µL/mL 2-mercaptoethanol (Wako), 2 ng/mL interleukin (IL)-3 (PeproTech, London, UK), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C under 5% CO2 and 95% air. The PB-3c cells (5 × 105 cells/mL) were precultured in culture media supplemented with 50 µM AA (Sigma-Aldrich) for 48 h. After washing the cells twice with PBS, 4 × 106 cells were placed in a microtube, resuspended in 180 µL Tyrode buffer containing the EP extracts, and incubated at 37°C for 10 min. Subsequently, the cells were stimulated with 10 µM calcium ionophore A23187 (Sigma-Aldrich) at 37°C for 20 min, and the stimulation was terminated by adding 200 µL acetonitrile/methanol (30/25, v/v) containing 1 mM ascorbic acid, 2 mM ethylenediaminetetraacetic acid (EDTA), and 1 µM prostaglandin (PG) B2 (Cayman Chemical, Ann Arbor, MI, USA) as an internal standard. The samples were stored at -80°C until HPLC analysis. The sample was centrifuged at 20 800 × g for 15 min at 4°C, and the supernatant was filtered. LTB4 in the supernatant was measured by HPLC on an ODS-A column (6.0 × 150 mm, 5 µm particle size, YMC, Kyoto, Japan) at 40°C. The injected samples (50 µL) were eluted with 5 mM CH3COONH4/acetonitrile/methanol (30/25/45, v/v/v) at a flow rate of 1.0 mL/min. LTB4 was detected by measuring the absorbance at 280 nm (SPD-10AVP, Shimadzu). Polyphenol determination Total polyphenol content in EP extracts was determined by the Folin–Ciocalteu method (Folin and Denis 1915). EP extracts were initially diluted with water. Then, 1 mL of the sample was mixed with 1 mL of 1 N Folin–Ciocalteu's reagent and incubated at 25°C for 3 min. Thereafter, 1 mL of 10% Na2CO3 (w/v) was added to the solution and incubated at 30°C for 30 min. The absorbance of the reaction mixture was measured at 760 nm. The results were calculated using a standard curve obtained from gallic acid under the same conditions and expressed as mg gallic acid equivalent (GAE). Chicoric acid in the extracts was determined by reversed-phase HPLC using an ODS column with UV detection according to a previously described method (Luo et al. 2003). Radical scavenging assay To determine the antioxidant properties of EP extracts, DPPH radical scavenging abilities were measured using Blois's method with some modifications (Blois 1958). The EP extracts were mixed with 100 µM DPPH in 75% ethanol and incubated for 30 min at 30°C in the dark. After centrifugation at 20 800 × g  at 20°C for 5 min, the absorbance of the reaction mixture was measured at 517 nm. The reaction mixture without samples and DPPH was used as a control and a blank, respectively. The radical scavenging ability was calculated as follows: $$\begin{eqnarray} &&\text{DPPH radical scavenging ability(%)}\\ &&\rm = {100 - (As - Ab)/(Ac - Acb) \times 100} \end{eqnarray}$$ where As is the absorbance of the sample, Ab is the absorbance of the blank, Ac is the absorbance of the control, and Acb is the absorbance of the control blank. Analysis of tyrosine-phosphorylated proteins RBL-2H3 cells on a 24-well culture plate were incubated with anti-DNP IgE for 20 h as described above. The IgE-sensitized cells were washed twice with PBS and suspended in 450 µL of Tyrode buffer containing 2.0 mg/mL of the petal extract. After incubation at 37°C for 10 min, 50 µL of DNP-BSA in Tyrode buffer was added and incubated for 20 min at 37°C. The reaction was terminated by washing the cells twice with cold Tris-buffered saline (TBS, pH 7.4) −1 mM EDTA. Cold lysis buffer (200 µL) consisting of TBS-EDTA with 1% Triton X-100, 50 mM NaF, and 5 mM Na3VO4 was added to the cells and incubated for 5 min on ice. The cell lysate was collected with a cell scraper and stored at -80°C until further analysis. Laemmli sample buffer with dithiothreitol (Atto, Tokyo, Japan) was added to the same amount of thawed sample and denatured at 95°C for 5 min. Proteins in the samples (30 µL) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an acrylamide slab gel system (AnykD TGX gel, Bio-Rad, Hercules, CA, USA) according to Laemmli's method (Laemmli 1970). Proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane by Western blotting. The PVDF membrane was incubated with Odyssey blocking buffer (TBS) provided by LI-COR (Lincoln, NE, USA) for 1 h. After washing with 0.05% Tween-TBS, the membrane was incubated with mouse monoclonal antibodies against phosphotyrosine (4G10, Millipore, Burlington, MA, USA) or β-actin (8H10D10, Cell Signaling Technology, Danvers, MA, USA) for 1 h. After washing with 0.05% Tween-TBS, the membrane was incubated with goat antimouse IgG conjugated with IRDye 800CW (LI-COR). The membrane used for detecting phosphotyrosine was treated with stripping buffer (LI-COR) to remove antibodies from the membrane according to the protocol of the provider. The membrane was reprobed with a primary antibody against Spleen tyrosine kinase (Syk, SYK-01, Thermo Fisher Scientific, Waltham, MA, USA) and goat antimouse IgG conjugated with IRDye 680RD (LI-COR) as a secondary antibody. The immunoreactive substances were analyzed by the Odyssey CLx (LI-COR) at near-infrared fluorescence (800 and 680 nm). Analysis of Ca2+ concentration in cytoplasm Ca2+ concentration in the cytoplasm of RBL-2H3 cells was measured using the Calcium Kit II-Fluo4 provided by Dojindo (Kumamoto, Japan). RBL-2H3 cells (3 × 104 cells/100 µL) were seeded in 96-well clear-bottom black microplates and incubated with anti-DNP IgE for 20 h. Further procedure was conducted according to the manufacturer's protocol. Briefly, RBL-2H3 cells sensitized with IgE were incubated with the loading buffer (5 µg/mL Fluo 4-AM, 0.04% Pluronic F-127, and 1.25 mM probenecid) in the presence of the petal extract (0.25, 0.5, and 1.0 mg/mL) at 37°C for 1 h. DNP-BSA was added to stimulate the cells. The fluorescence intensity (excitation at 485 nm and emission at 520 nm) was continuously measured using a fluorescence plate reader (PerkinElmer WALLAC 1420 ARVOmx/Light, Waltham, MA, USA) at 37°C. Statistical analysis Data are expressed as the mean ± SD (n = 3). The experiments were conducted several times to confirm the reproducibility of the results. Statistical significance of differences was analyzed using the Tukey–Kramer multiple comparison test. Differences with P values less than .05 were considered significant. Results Effect of EP extracts on histamine release from RBL-2H3 Inhibitory activities of the EP extracts (1.5 mg/mL) against the release of histamine from RBL-2H3 stimulated by antigen-IgE cross-linking were evaluated (Figure 1). The petal extract significantly suppressed histamine release, which was 16% of the histamine release in the absence of the EP extract (control). The leaf extract appeared to suppress the release of histamine, although the activity was not so strong as the petal extract. On the other hand, the stem extract did not affect the release of histamine. Chicoric acid, a major polyphenol in EP (Molgaard et al. 2003), did not exert significant inhibitory effect on the histamine release at 10 µg/mL (data not shown). The EP extracts did not exert cytotoxicity at a concentration of 2.0 mg/mL as determined by the Trypan blue assay (data not shown). Figure 1. Open in new tabDownload slide Effect of EP extracts on histamine release from RBL-2H3. RBL-2H3 cells were precultured with anti-2,4-DNP IgE for 20 h, and the cells were stimulated with DNP-BSA for 20 min at 37°C in the presence of EP extracts. The histamine in the supernatant was determined by HPLC with FL detection. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 1. Open in new tabDownload slide Effect of EP extracts on histamine release from RBL-2H3. RBL-2H3 cells were precultured with anti-2,4-DNP IgE for 20 h, and the cells were stimulated with DNP-BSA for 20 min at 37°C in the presence of EP extracts. The histamine in the supernatant was determined by HPLC with FL detection. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 2 shows the effect of the petal extract on the release of histamine from RBL-2H3 cells at various concentrations. A dose-dependent tendency was observed at 0.5-2.0 mg/mL of petal extract, and the suppressive effect at 1.0 and 2.0 mg/mL were significant (P < .05). Figure 2. Open in new tabDownload slide Dose-dependent suppressive effect of EP petal extract on histamine release from RBL-2H3. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 2. Open in new tabDownload slide Dose-dependent suppressive effect of EP petal extract on histamine release from RBL-2H3. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Effect of EP extracts on LTB4 production in PB-3c Inhibitory activities of the EP extracts (1.5 mg/mL) against LTB4 production in PB-3c cells stimulated by calcium ionophore were evaluated (Figure 3). All the extracts significantly reduced LTB4 production to approximately 50% compared with control. Although statistical differences in the inhibitory activities were not observed among the EP extracts, the inhibitory effect of the petal extract tended to be stronger than that of leaf and stem extracts. Chicoric acid did not exert significant inhibitory effect on the LTB4 production at 10 µg/mL (data not shown). None of the EP extracts showed cytotoxicity at 2.0 mg/mL as determined by the Trypan blue assay (data not shown). Figure 3. Open in new tabDownload slide Effect of EP extracts on LTB4 production in PB-3c. PB-3c was precultured with AA for 48 h, and the cells were stimulated with calcium ionophore for 20 min. LTB4 in the cell lysate was determined by HPLC with UV detection. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 3. Open in new tabDownload slide Effect of EP extracts on LTB4 production in PB-3c. PB-3c was precultured with AA for 48 h, and the cells were stimulated with calcium ionophore for 20 min. LTB4 in the cell lysate was determined by HPLC with UV detection. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 4 indicates the effect of the petal extract on LTB4 production in PB-3c at various concentrations. Significant dose-dependent activities were demonstrated at 1.0-2.0 mg/mL of petal extract (P < .05). Figure 4. Open in new tabDownload slide Dose-dependent suppressive effect of EP petal extract on LTB4 production in PB-3c. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 4. Open in new tabDownload slide Dose-dependent suppressive effect of EP petal extract on LTB4 production in PB-3c. Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Polyphenol contents of EP extracts Figure 5 shows the total polyphenol content of the EP extracts. The average amounts of petal, leaf, and stem extracts were 73.5, 33.1, and 15.4 mg GAE/g dried extract, respectively. Chicoric acid in petal, leaf, and stem extracts were 3.23, 4.58, and 1.77 mg/g dried extract, respectively. Figure 5. Open in new tabDownload slide Polyphenol contents of EP extracts. The sample was mixed with Folin–Ciocalteu's reagent and incubated at 25°C for 3 min. After incubation with Na2CO3 at 30°C for 30 min, the absorbance at 760 nm was measured. The amounts were calculated in terms of mg gallic acid equivalent (GAE). Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Figure 5. Open in new tabDownload slide Polyphenol contents of EP extracts. The sample was mixed with Folin–Ciocalteu's reagent and incubated at 25°C for 3 min. After incubation with Na2CO3 at 30°C for 30 min, the absorbance at 760 nm was measured. The amounts were calculated in terms of mg gallic acid equivalent (GAE). Data represent the mean ± SD (n = 3). Means without a common letter are significantly different (P < .05). Radical scavenging ability of EP extracts Figure 6 shows the DPPH radical scavenging abilities of EP extracts. All the extracts exhibited dose-dependent scavenging abilities. The half-maximal inhibitory concentrations (IC50) of petal, leaf, and stem extracts were 24.9, 58.8, and 159.2 µg/mL, respectively. Figure 6. Open in new tabDownload slide Radical scavenging ability of EP extracts. EP extracts were mixed with DPPH in ethanol/water and incubated for 30 min at 30°C in the dark. The absorbance of the reaction mixture at 517 nm was measured. Data represent the mean ± SD (n = 3). Petal extract (●), leaf extract (■), and stem extract (▲). Figure 6. Open in new tabDownload slide Radical scavenging ability of EP extracts. EP extracts were mixed with DPPH in ethanol/water and incubated for 30 min at 30°C in the dark. The absorbance of the reaction mixture at 517 nm was measured. Data represent the mean ± SD (n = 3). Petal extract (●), leaf extract (■), and stem extract (▲). Effect of EP petal extract on tyrosine phosphorylation of proteins Western blot analysis using primary antiphosphotyrosine (top left panel) and anti-β-actin antibody (bottom left panel) for RBL-2H3 cell lysate following stimulation by antigen-IgE cross-linking in the presence of the EP petal extract was illustrated in Figure 7. As compared to no stimulation (lane 1), an increase in phosphotyrosine residues of the cell proteins with a molecular weight of around 70 kDa (upper arrow) and 30 kDa (lower arrow) was observed (lane 2). On the other hand, 2.0 mg/mL of the petal extract suppressed the tyrosine phosphorylation (lane 3). The bands at 70 kDa were identified as Syk (middle left panel) by the detection using anti-Syk antibody after the stripping. There was no difference in Syk levels among no stimulation (lane 1), control (lane 2), and petal extract (lane 3). The fluorescence intensities of the bands at 70 kDa detected by antiphosphotyrosine antibody were quantified and the values were normalized by each fluorescence intensity of Syk band (top right panel). The suppression of tyrosine phosphorylation at 70 kDa by the petal extract was significant (P < .05). There was no difference in β-actin levels at 42 kDa, a loading control of total cell proteins, among no stimulation (lane 1), control (lane 2), and petal extract (lane 3). Figure 7. Open in new tabDownload slide Effect of EP petal extract on tyrosine phosphorylation. IgE-sensitized RBL-2H3 cells were stimulated with DNP-BSA for 20 min at 37°C in the presence of EP extracts. The cells were treated with lysis buffer, and the cell lysates were subjected to SDS-PAGE followed by Western blotting using primary antiphosphotyrosine (top left panel), Syk (middle left panel), or anti-β-actin (bottom left panel) antibodies. Lane 1, no stimulation; lane 2, control; lane 3, petal extract (2.0 mg/mL). The fluorescence intensities of Western blot bands at 70 kDa detected by antiphosphotyrosine were quantified and the values were normalized by each Syk content (top right panel). Results are shown as mean ± SD of 3 independent experiments. Means without a common letter are significantly different (P < .05). Figure 7. Open in new tabDownload slide Effect of EP petal extract on tyrosine phosphorylation. IgE-sensitized RBL-2H3 cells were stimulated with DNP-BSA for 20 min at 37°C in the presence of EP extracts. The cells were treated with lysis buffer, and the cell lysates were subjected to SDS-PAGE followed by Western blotting using primary antiphosphotyrosine (top left panel), Syk (middle left panel), or anti-β-actin (bottom left panel) antibodies. Lane 1, no stimulation; lane 2, control; lane 3, petal extract (2.0 mg/mL). The fluorescence intensities of Western blot bands at 70 kDa detected by antiphosphotyrosine were quantified and the values were normalized by each Syk content (top right panel). Results are shown as mean ± SD of 3 independent experiments. Means without a common letter are significantly different (P < .05). Effect of EP petal extract on cytoplasmic Ca2+ concentration Figure 8 shows the time-course analysis of cytoplasmic Ca2+ concentration in RBL-2H3 stimulated by antigen-IgE cross-linking in the presence of EP petal extract. The relative fluorescence intensity of the control was increased after stimulation by adding DNP-BSA, whereas the intensity was constant without stimulation. The relative fluorescence intensity was dose-dependently suppressed by the petal extract, in particular, 1.0 mg/mL of the petal extract exhibited strong inhibition of calcium influx. Figure 8. Open in new tabDownload slide Effect of EP petal extract on cytoplasmic Ca2+ concentration. RBL-2H3 cells sensitized with IgE were incubated with a fluorescent probe at 37°C for 1 h in the presence of petal extract. The cells were stimulated by adding DNP-BSA (antigen, ⟶). The fluorescence intensity was continuously monitored. Control (●), 0.25 mg/mL (■), 0.5 mg/mL (▲), 1.0 mg/mL (◆), and no stimulation (○). Figure 8. Open in new tabDownload slide Effect of EP petal extract on cytoplasmic Ca2+ concentration. RBL-2H3 cells sensitized with IgE were incubated with a fluorescent probe at 37°C for 1 h in the presence of petal extract. The cells were stimulated by adding DNP-BSA (antigen, ⟶). The fluorescence intensity was continuously monitored. Control (●), 0.25 mg/mL (■), 0.5 mg/mL (▲), 1.0 mg/mL (◆), and no stimulation (○). Discussion Suppression of the release of chemical mediators from basophilic leukocytes and mast cells is key to relieving the type-I allergic symptoms such as mucus hypersecretion. Antiallergic drugs are designed to inhibit chemical mediators, although they cause some side effects such as drowsiness. This has led to the search for antiallergic natural compounds without side effects, such as polyphenols, which have been identified in various foods (Singh, Holvoet and Mercenier 2011). Echinacea has traditionally been used for herbal tea with safety food experiences and has been reported to have anti-inflammatory effects (Borchers et al. 2000). Gulledge et al. (2018) have reported the antiallergic effects of Echinacea roots, suggesting that dodeca-2E,4E-dienoic acid isobutylamide is an active component. The objective of this study was to elucidate the antiallergic effects of the aerial part of EP using cultured cell lines, because the aerial part of the plant is also a major ingredient besides the root for herbal tea. We prepared the extracts from the aerial part of EP divided into petals, leaves, and stems using hot water as is done in making herbal tea. It has been reported that the amounts of alkylamides in EP aerial parts are very low as compared to the roots (Molgaard et al. 2003). Accordingly, the extracts may contain hydrophilic substances such as polyphenols, although the amounts of hydrophobic substances such as alkylamides could be low in the extracts. Histamine is a chemical mediator that is released by degranulation as a result of the cross-linking of IgEs with allergens on the surface of mast cells and basophilic leukocytes (Siraganian 2003). The inhibitory activity of the EP extracts against the release of histamine was evaluated on a rat basophilic leukemia cell line using a method previously described with some modifications (Matsuo et al. 1997; Byeon et al. 2009). Our data in Figures 1 and 2 demonstrate that the petal extract significantly inhibits the release of histamine as compared to leaf and stem extracts. It is necessary to identify the responsible components in the hot water extracts of EP for this inhibitory activity. It has been suggested that polyphenols present in plants and foods can suppress the release of chemical mediators from mast cells (Singh, Holvoet and Mercenier 2011; Maeda-Yamamoto 2013; Kumazawa et al. 2014). In the present study, a positive correlation was observed between the inhibitory activity against the release of histamine (Figure 1) and the total polyphenol content (Figure 5) of the extracts. Thus, the inhibitory effect of the petal extract on the release of histamine may be due to polyphenols, although the exact chemical compound is not clear. Chicory acid has been reported as a major polyphenol in EP, especially abundant in the leaves (Molgaard et al. 2003). We have determined the chicoric acid in the EP water extracts and examined the inhibitory effect on the histamine release from RBL-2H3 at 10 µg/mL, which corresponds to more than 2 mg/mL of the extracts, however, chicoric acid did not affect the histamine release. This means that other polyphenols or hydrophilic compounds in the extract may inhibit the histamine release. Further experiments are needed to identify hydrophilic components other than chicoric acid involved in the inhibition of histamine release. LTs are another type of chemical mediators produced through a pathway of the AA cascade mediated by 5-LOX in mast cells after stimulation by antigens (Duroudier, Tulah and Sayers 2009). The LOX reaction is a type of lipid peroxidation, in which lipid radicals are generated as the intermediate substances (Schneider et al. 2007). It has been suggested that antioxidants such as polyphenols can inhibit the inflammatory LOX reaction (Santangelo et al. 2007; Yahfoufi et al. 2018). We have developed an experimental method to evaluate the inhibitory activity of food components such as polyphenols against LTB4 production using a mast cell line (Takasugi et al. 2018), to which an assay was applied for the evaluation of the EP extracts. As shown in Figure 3, the petal, leaf, and stem extracts have similar activities in suppressing the LTB4 production, in which the activity of petal extract seems to be stronger than the others, while their polyphenol amounts are not the same (Figure 5). The significant inhibitory effect of chicoric acid at 10 µg/mL corresponding to more than 2 mg/mL of the extracts on the LTB4 production in PB-3c was not observed. Based on these results, we hypothesized that antioxidants other than polyphenols might be associated with the inhibition of LTB4 production by leaf and stem extracts, and therefore, the antioxidant activity of the extracts was evaluated as DPPH radical scavenging ability, which is a widely used method to evaluate the antioxidant activity of food components. Results showed that the antioxidant activity of the petal extract was the strongest, followed by the leaf and stem extracts (Figure 6), indicating that there is a positive correlation between the polyphenol content and antioxidant activity of the extracts. This means that the antioxidant activities of the extracts are likely to be derived from polyphenols in the extracts. Consequently, any water-soluble substances other than antioxidants in the leaf and stem extracts could be involved in their ability to inhibit LTB4. After IgE-antigen stimulation, mast cells induce tyrosine phosphorylation of signaling molecules via a cascade reaction, followed by Ca2+ influx into the cytoplasm, which leads to the release of chemical mediators (Siraganian 2003). We focused on the petal extract because it exerted the strongest inhibitory activity on the release histamine as well on LTB4 production, and we examined the effect on signal transduction in the stimulated mast cell line to elucidate the mechanisms underlying the inhibition of chemical mediators. The petal extract suppressed tyrosine phosphorylation of the signal molecule with a size of approximately 70 kDa, as illustrated in Figure 7. Syk is a 72 kDa-signaling molecule which mainly binds to the gamma chain of FcεRI, and the interaction results in its activation and phosphorylation, which in turn phosphorylate downstream proteins (Gilfillan and Rivera 2009; Siraganian et al. 2010). The signal molecule around 70 kDa was confirmed as Syk by reprobing the same membrane with a specific antibody to Syk. The EP petal extract also inhibited intracellular Ca2+ influx, as indicated in Figure 8, which may be due to the inhibition of upstream tyrosine phosphorylation, although more detailed mechanisms need to be elucidated. These data indicate that the EP petal extract can suppress histamine release and LTB4 production, in which the inhibition of signal transduction of Syk and Ca2+ influx may be associated. Further studies will be required to clearly understand the uptake of the EP extracts into cells and their activity in vivo using allergic animal models. In conclusion, these data suggest that the hot water extract of EP petals may alleviate symptoms of type-I allergies by inhibiting the release of chemical mediators from basophilic leukocytes and mast cells. Acknowledgments The authors are grateful to Mizuki Nakamura (Kitami Institute of Technology) for technical assistance. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Author contribution H.A. designed this study and prepared the materials. A.Z., R.T., and E.S. performed the experiments. H.A., A.Z., M.T., and R.T. analyzed the data and discussed the results. A.Z., H.A., and M.T. wrote the manuscript. Funding None declared. Disclosure statement No potential conflict of interest was reported by the authors. References Amin K . The role of mast cells in allergic inflammation . Respir Med 2012 ; 106 : 9 - 14 . 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TI - Echinacea purpurea water extracts suppress the release of chemical mediators from mast cells
JO - Bioscience Biotechnology and Biochemistry
DO - 10.1093/bbb/zbaa125
DA - 2021-01-06
UR - https://www.deepdyve.com/lp/oxford-university-press/echinacea-purpurea-water-extracts-suppress-the-release-of-chemical-Av7Js1rMrv
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -