Introduction
Adaptive immunity serves the important function of host defense against microbial infections, but immune responses are also capable of causing tissue injury and disease. Disorders caused by immune reponses are called hypersensitivity (Abbas et al., 2012). The term ‘allergy’ was coined by Clemens von Pirquet in 1906 to call attention to the unusual propensity of some individuals to develop signs and symptoms of reactivity, or ‘hypersensitivity reactions’ when exposed to certain substances. Allergic disorders are also associated with the production of allergen-specific IgE and with the expansion of allergen-specific T-cell populations, both of which are reactive with what typically are otherwise harmless environmental substances (Galli et al., 2008).
Immediate hypersensitivity (Type I allergy) diseases, commonly called allergic or atopic disorders, are the prototypes of diseases caused by activation of the TH2 subset of helper T cells, in which the T cells stimulate the production of IgE antibodies and inflammation (Abbas et al., 2012). Type I allergy is an immune disorder that involves the production of immunoglobulin E (IgE) in response to allergens and antigens. Antigen-induced release of inflammatory mediators from mast cells cause the immediate symptoms of IgE-mediated allergic diseases, including allergic rhinitis, asthma, atopic dermatitis, and atopic eczema (Han et al., 2009).
Mast cells and basophils not only are important effector cells in acute IgE-associated allergic reactions but also may contribute significantly to the expression of aspects of acquired immune responses that develop over hours or days to weeks. Mast cells and basophils may also mediated immunoregulatory functions, both through their ability to produce certain cytokines and by other mechanisms (Wedmeyer et al., 2000).
While it is currently estimated that about 25% of the population of the industrialized world suffers from allergy of various forms (Cheng et al., 2013), and the prevalence of childhood atopic dermatitis in low-income countries is on increase (Flohr & Mann, 2014).
Recently, studies on anti-allergy or anti-degranulation effect of plants such as Pueraiae Radix, fermented red ginseng, red ginseng and ginseng were reported (Jeong et al., 2013; Park et al., 2010; Park et al., 2011).
This study was conducted to screen plants with inhibitory activity on the degranulation-induced mast cells, which could be expected to use for anti-allergy resources.
Materials and Methods
Plant extract samples
Fifty plant extracts including of the methanol extract from the aerial part of Allium tuberosum Rottler ex Spreng. were listed in Table 1, in which the information such as the places collected, the plant parts used, the extracting conditions and the time collected were listed. The extracts were distributed from Plant Extract Bank of National Institute of Hoticultural and Herbal Science (NIHHS).
Table 1.z1, Yanggu; 2, Odaesan; 3, Taean; 4, Geumwonsan; 5, Eumsung (RDA); 6, Incheon; 7, Eumsung; 8, Suwon (RDA); 9, Sobaeksan; 10, Yeongju; 11, Suncheon; 12, Songnisan.yThe extracting solvents and conditions; A, methanol at 50℃ by accelerated solvent system; B, ethanol at room temperature; C, water at room temperature; D, methanol at room temperature.
Cell lines and culture
The mast cells, RBL-2H3 cell line were obtained from Korean Cell Line Bank (KCLB). Before use for assay, the mast cells were plated at 1.25×105 cells/well in 48 well culture plate which contains Minimum Essential Medium Eagle (MEM) media supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml Aupho B, and cultured at 37℃ and 5% CO2 atmosphere condition.
Analysis for cytokine (interleukin-4) release
The assay of interleukin (IL)-4 production was conducted by the manual suggested by Abcam as below. Each standard and sample (100 μl) were added into wells, covered and incubated for 2.5 hours at room temperature with gentle shaking. The solution was discarded and each well was washed 4 times. After the last wash, remaining wash buffer was removed by aspirating. Added 100 μl of 1× biotinylated IL-13 detection antibody to each well. Incubated for 1 hour at room temperature with gentle shaking. Discarded the solution. Repeated wash procedure. Added 100 μl of 1× HRP-streptavidin solution to each well. Incubated for 45 minutes at room temperature with gentle shaking. Discarded the solution. Repeated wash, and added 100 μl of TMB substrate reagent to each well. Incubated for 30 minutes at room temperature in the dark with gentle shaking. Added 50 μl of stop solution to each well. Read at 450 nm immediately.
Analysis of β-hexosaminidase release
The media of the cultured RBL-2H3 cells were exchanged with Tyrode’s buffer, and incubated with 200 ng/ml DNP-IgE (Sigma-Aldrich, St. Louis, MO, USA) at 37℃ and 5% CO2 conditions for 24 hours. At one hour after treating of the plant extract, cells were added with 50 ng/ml DNP-BSA (Sigma-Aldrich, St. Louis, MO, USA), were cultured for 2 hours, and were stopped the degranulation on the ice bath. For the assay on β-hexosaminidase release, the volume (25 μl) of cell supernatant was transferred to 96 well black plate, and was mixed with 100 μl of 2.4 mM 4-methylumbelliferyl-N-acetyl-b-D-glucosaminide (Sigma-Aldrich, St. Louis, MO, USA) and finally was incubated for 1 hour. The reaction was stopped via addition with 175 μl of 0.1 M glycine-carbonate buffer (pH 10.0), and its fluorescence at 360nm/450nm was analyzed in the plate reader.
Cytotoxicity test
The cultured cells were treated with plant extracts at the final concentrations at 100 μg/ml for 24 hours, and treated with dimethyl sulfoxide (DMSO) as negative control. The cells were incubated and treated with 200 μg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, USB, Cleveland, Ohio, USA) for 3 hours at 37℃ and 5% CO2 incubator. After MTT reagent was eliminated and 100 μl DMSO was added, the optical density of the DMSO solution was analyzed at 550 nm by using plate reader (Biotek, Synergy HT, Winooski, VT, USA).
Statistical analysis
The results (n = 2~3) were showed as average ± standard error and the statistical significance was determined by Student’s t-test (p < 0.05).
Results and Discussion
Effect of plant extracts on IL-4 release
The effects of plant extracts at 20 μg/ml concentration were analyzed on the release of IL-4 of IgE-sensitized RBL-2H3 cells. From the experiment, the plant extracts with above 30% suppressive effect on the IL-4 production included 39 extracts including Cardamine impatiens L. (aerial part, methanol extract), Magnolia sieboldii K. Koch (aerial part, methanol extract), Robinia pseudoacacia L. (branch, methanol extract), Hydrangea serrata for. acuminata (Siebold & Zucc.) Wilson (aerial part, methanol extract), Mentha piperascens (Malinv.) Holmes (aerial part, ethanol extract), Asarum sieboldii Miq. (root, methanol extract), Nelumbo nucifera Gaertn. (stamen/ovary, methanol extract), Nelumbo nucifera Gaertn. (petal, methanol extract), Sorbus alnifolia (Siebold & Zucc.) K. Koch (leaf, methanol extract), Paeonia suffruticosa Andrews (root peel, water extract), Gomphrena globosa L. (root, methanol extract), Rhus javanica L. (aerial part, methanol extract), Hibiscus mutabilis L. (stem, methanol extract), Quercus mongolica Fisch. ex Ledeb. (leaf/stem, methanol extract), etc. Among the samples, the methanol extract of Cardamine impatiens L. (aerial part), Magnolia sieboldii K. Koch (aerial part), Robinia pseudoacacia L. (branch), Hydrangea serrata for. acuminata (Siebold & Zucc.) Wilson (aerial part), and the ethanol extract of Mentha piperascens (Malinv.) Holmes (aerial part) indicated high suppressive efficacies on IL-4 release as 89.8 ± 0.7%, 83.5 ± 1.3%, 79.0 ± 6.5%, 73.0 ± 4.3% and 70.3 ± 0.4%, respectively (Table 2).
Table 2.zFinal concentration of sample for the evaluation for IL-4 assay was 20 μg/ml.ySymbol indicates significance of the data, p < 0.05.
Effect of plant extracts on β-hexosaminidase release
For evaluating the effects of plant extracts on the degranulation-induced mast cells (RBL-2H3 cell line), the inhibition activity on release of β-hexosaminidase was analyzed. From the assay, the extracts at 20 μg/ml concentration showing above 30% inhibitory activity were 23 samples including Lindera erythrocarpa Makino (aerial part, methanol extract), Geum japonicum Thunb. (leaf/stem, methanol extract), Spiraea prunifolia for. simpliciflora Nakai (leaf, methanol extract), Magnolia sieboldii K. Koch (aerial part, methanol extract), Weigela subsessilis (Nakai) L. H. Bailey (leaf, methanol extract), Opuntia ficus-indica Mill. (leaf, methanol extract), Nelumbo nucifera Gaertn. (leaf, methanol extract), Rumex acetosella L. (aerial part, methanol extract), Artemisia capillaris Thunb. (root, methanol extract), Lindera obtusiloba Blume (aerial part, methanol extract), etc. Among this extracts, the methanol extracts of Lindera erythrocarpa Makino (aerial part), Geum japonicum Thunb. (lea/stem), Spiraea prunifolia for. simpliciflora Nakai (leaf) showed effective inhibition activity as 87.2 ± 0.7%, 77.5 ± 1.5% and 71.2 ± 1.3% on β-hexosaminidase release, respectively (Table 3).
Table 3.zFinal concentration of sample for the evaluation for β-hexosaminidase was 20 μg/ml.ySymbol indicates significance of the data, p < 0.05.
Effects of plant extract on cell proliferation
Fifty samples were estimated the cytotoxicity on RBL-2H3 cells via MTT assay and the results are shown at Table 4. At 100 μg/ml concentration, the extracts indicated above 80% in cell proliferation were 26 including methanol extract of Weigela subsessilis (Nakai) L. H. Bailey (branch), and the extracts indicated below 50% proliferation were 13 including the methanol extract of Sorbaria sorbifolia var. stellipila Maxim. (leaf/branch). Among the plant extracts, Weigela subsessilis L.H.Bailey (branch, methanol extract), Rhus javanica L. (aerial part, methanol extract), Paeonia suffruticosa Andrew. (root peel, ethanol extract), Paeonia lactiflora Pall. var hottensis Makino (root, ethanol extract), Caryopteris incana (Thunb.) Miq. (aerial part, methanol extract), Paeonia suffruticosa Andrew. (root peel, water extract) showed very high values in cell viabilities as 136.8 ± 1.5%, 132.1 ± 1.2%, 112.4 ± 1.4%, 111.6 ± 8.1%, 101.2 ± 2.6%, and 100.6 ± 5.5%, respectively.
Table 4.zFinal concentration of sample for the evaluation of cellular cytotoxicity was 100 μg/ml.yDimethyl sulfoxide (DMSO) was used as negative control which was not treated with DNP-IgE, DNP-BSA and plant extract.xSymbol indicates significance of the data, p < 0.05.
From the results, six plant extracts such as Geum japonicum Thunb. (leaf/stem), Nelumbo nucifera Gaertn. (stamen/ovary), Nelumbo nucifera Gaertn. (stem), Opuntia ficus-indica Mill. (leaf), Paeonia lactiflora Pall. var hottensis Makino (root) and Rhus javanica L. (aerial part), were preliminarily selected as useful plant samples. This result was evoked from the data on three bio-markers such as the inhibition of IL-4 release, β-hexosaminidase release and cell viability shown before (at Table 2, Table 3, and Table 4). In vitro anti-degranulation effects of these plants at other concentration in the aspects of IL-4 release, β-hexosaminidase release and cell proliferation were analyzed further, and the results were indicated at Fig. 1, Fig. 2 and Fig. 3.
Fig. 1.Inhibition effect of the plant extracts of Geum japonicum Thunb. (leaf/stem, sample no.15), Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 26), Nelumbo nucifera Gaertn. (stem, sample no. 28), Opuntia ficus-indica Mill. (leaf, sample no. 29), Paeonia lactiflora Pall. var hottensis Makino (root, sample no. 30) and Rhus javanica L. (aerial part, sample no. 36) on IL-4 release in IgE-sensitized RBL-2H3 cells.
Fig. 2.Inhibition effect of the plant extracts of Geum japonicum Thunb. (leaf/stem, sample no.15), Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 26), Nelumbo nucifera Gaertn. (stem, sample no. 28), Opuntia ficus-indica Mill. (leaf, sample no. 29), Paeonia lactiflora Pall. var hottensis Makino (root, sample no. 30) and Rhus javanica L. (aerial part, sample no. 36) on β-hexosaminidase release in IgE-sensitized RBL-2H3 cells.
Fig. 3.Cell viability of the plant extracts of Geum japonicum Thunb. (leaf/stem, sample no.15), Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 26), Nelumbo nucifera Gaertn. (stem, sample no. 28), Opuntia ficus-indica Mill. (leaf, sample no. 29), Paeonia lactiflora Pall. var hottensis Makino (root, sample no. 30) and Rhus javanica L. (aerial part, sample no. 36) in IgE-sensitized RBL-2H3 cells.
Among the plants, inhibition effects of the plant extracts of Geum japonicum Thunb. (leaf/stem), Nelumbo nucifera Gaertn. (stamen/ovary) and Paeonia lactiflora Pall. var hottensis Makino (root, ethanol extract) on IL-4 release increased on concentration dependently and the stamen/ovary extract of Nelumbo nucifera Gaertn. showed the most effective activity as 59.2 ± 1.2% and 96.4 ± 0.4% at 20 and 100 μg/ml, respectively (Fig. 1).
On β-hexosamnidase release in IgE-sensitized RBL-2H3 cells, Geum japonicum Thunb. (leaf/stem), Nelumbo nucifera Gaertn. (stamen/ovary), and Rhus javanica L. (aerial part) inhibited in dose-dependent mode, and Geum japonicum Thunb. (leaf/stem) indicated the most suppressive capacity as 77.5 ± 1.5% and 81.6 ± 7.9% at 20 and 100 μg/ml, respectively (Fig. 2).
In cell viability of the plant extracts in IgE-sensitized RBL-2H3 cells, Geum japonicum Thunb. (leaf/stem), Paeonia lactiflora Pall. var hottensis Makino (root, ethanol extract) and Rhus javanica L. (aerial part) indicated higher values than other plants as 95.4 ± 1.0~104.5 ± 1.4%, 96.1 ± 5.4~111.6 ± 8.1% and 95.3 ± 1.6∼132.1 ± 1.2%.
Finally, the results provided that the extracts from leaf/stem of Geum japonicum Thunb. and stamen/ovary of Nelumbo nucifera Gaertn. showed higher effective activities in the inhibition on β-hexosaminidase release and IL-4 production and did high cell proliferation activities.
The study was conducted to screen plant resources having anti-allergy activity in a species of mast cells, that is rat basophilic leukemia cells (RBL-2H3 cells). Mast cells arise from pluripotent stem sells, mature in tissue and have the ability to generate inflammation following exposure to a variety of receptor-mediated signals initiated by both innate and acquired immune response mechanisms (Brown et al., 2007). Mast cells express receptors (FcεRI) that bind immunoglobulin (Ig) E antibodies with high affinity, and aggregation of these FcεRI by the reaction of cell-bound IgE with specific antigens induces mast cells to secrete a broad spectrum of biologically active preformed or lipid mediators, as well as many cytokines. So mast cells are widely thought to be essential for the expression of acute allergic reactions, but the importance of mast cells in late-phase reactions and chronic allergic inflammation has remained controversial (Williams & Galli, 2000).
Allergy begins with allergen sensitization, which elicit a TH2 immune reponses such as an increase IL-4 and IL-13 levels, leading to immunoglobulin class switching B cells to IgE predominant production (Galli et al., 2008). Re-exposure to allergen triggers cross-linking of allergen-specific IgE-bound high-affinity IgE receptors (FcεRI) on mast cells and basophils, stimulation of upstream Syk tyrosine kinase and downstream phospholipase Cγ (PLCγ), leading to an increase in intracellular inositol (1,4,5)-triphosphate (IP3) and Ca2++ levels and degranulation and release of mediators (Cheng et al., 2013). Increase in cytosolic Ca2++ levels is the single most critical cellular signal for mast cell degranulation (Nishida et al., 2005).
Upon activation of mast cells via crosslinking of the high affinity IgE receptor (FcεRI) or non-mediated activation through complement receptors or toll-like receptor (TLP) activation, the activated mast cells can release a variable spectrum of pro-inflammatory mediators. These include mediators such as histamine, serotonin and proteases, leukotrienes and prostaglandins and cytokines and chemokines. The levels and pattern of mediator release is influenced by cytokines, growth factors and microenvironmental conditions. IL-4 enhances FcεRI-mediated reactions from human mast cells (Brown et al., 2007). IL-4 is essential for IgE production and promotes the differentiation of naive T cells into TH2 cells (Han et al., 2011). After stimulation with antigen, cells release β-hexosaminidase, which is a marker of mast cell degranulation, and various allergic mediators including histamine, cytokines and arachidonic acid derivatives (Han et al., 2009).
For verifying in vivo anti-allergy capacity of selected candidates, animal models including passive cutaneous anaphylaxis (PCA), active cutaneous anaphylaxis (ACA), atopic dermatitis and/or asthma model by using experimental animal such as mice etc, could be used (Megumi et al., 2012; Shimada et al., 2004; Choi et al., 2011; Shimizu et al., 2012). Lim et al. (2011) reported the results on the epidermal recovering-effects of the Cnidium officinale extract after inducing contact-dermatitis by 1-chloro-2,4-dinitrochlorobenzene (DNCB) in Sprague-Dawley rats.
In the study, we experimented anti-degranulation activity of 50 plant extracts by measuring the amounts of β-hexosaminidase, and IL-4, and analyzing cell viability. From the analysis, six plant samples were preliminarily selected as useful anti-allergy materials, which were tested in other concentration further. Considering the results about the suppressive activities on IL-4 production, β-hexosaminidase release in the degranulation-induced mast cells and the cell viabilities, the leaf/stem of Geum japonicum Thunb. and the stamen/ovary of Nelumbo nucifera Gaertn. could be useful resources for anti-degranulation materials. And these two plant parts are expected to be utilized as efficient material for treatment and/or amelioration of allergy reaction, and thus need to be studied for the utilization.
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