Anti-Allergic Effects of Lonicera caerulea L. Extract and Cyanidin-3-Glucoside on Degranulation and FcεRI Signaling Pathway of RBL-2H3 Cells

Abstract

(i) Background: The increasing prevalence of allergic diseases highlights the need for effective treatments. Lonicera caerulea fruit has been recognized for its anti-inflammatory, anti-cancer, and neuroprotective effects, but the mechanisms underlying its anti-allergic properties remain unclear. (ii) Objective: This study aims to evaluate the total phenolic, total flavonoid, and cyanidin-3-glucoside (C3G) contents of Lonicera caerulea extract (HR2302-30E) and to investigate its antioxidant and anti-allergic activities. (iii) Methods: Using an IgE-stimulated RBL-2H3 cell model, we assessed the effects of HR2302-30E and C3G on mast cell degranulation, β-hexosaminidase and histamine release. Western blot analysis was performed to evaluate the expression of high-affinity IgE receptor (FcεRI)β/γ and the phosphorylation of Src family kinases (Syk, Fyn). We also examined the phosphorylation of downstream factors phospholipase Cγ, protein kinase Cδ, and mitogen-activated protein kinase. (iv) Results: Total phenolic, flavonoid, and C3G contents of HR2302-30E were 18.73 mg GAE/g, 11.83 mg QE/g, and 7.02 mg/g, respectively. In IgE-activated mast cells, HR2302-30E and C3G inhibited β-hexosaminidase and histamine release. Western blot analysis revealed reduced expression of FcεRIβ/γ and decreased phosphorylation of key downstream signaling molecules. Conclusions: These findings suggest that HR2302-30E and C3G modulate FcεRI signaling, indicating their potential as natural anti-allergic agents.

1. Introduction

The prevalence of allergic diseases is increasing worldwide, affecting approximately 10–30% of the global population [1]. Allergic diseases result from immune system dysfunction and encompass conditions such as atopic dermatitis (AD), allergic asthma (AAS), allergic rhinitis (AR), and food allergies (FAs). For example, the annual prevalence of atopic diseases is 1.2% in adults in Asia and 17.1% in Europe, while the prevalence among children in Asia ranges from 0.96% to 22.6% [2]. In Japan, the prevalence of allergic diseases among adolescents and adults (ages 18–50) is as follows: AD 15.6%, AAS 14.7%, AR 47.4%, and FAs 12.3%, with AR being the most common [3]. In South Korea, the prevalence of allergic diseases in children is as follows: for infants (>2 years old), AD, AAS, and AR are 9.0%, 0.9%, and 5.9%, respectively; for preschool children (2–5 years old), 20.2%, 2.3%, and 11.3%; and for school-age children (6–18 years old), 27.6%, 4.1%, and 14.6%. In adults, the prevalence is 17.1%, 3.9%, and 2.3%, respectively, with lower rates observed in the elderly (6.9%, 4.1%, 1.6%) [4].

Allergic diseases manifest in various parts of the body, leading to diverse clinical symptoms [5]. AD is caused by skin barrier dysfunction, abnormal skin microbiota, and localized Th2 immune responses, leading to symptoms such as itching, dryness, and skin lesions [6]. AR and AAS are triggered by allergens, such as airborne molds and pollen. AR leads to symptoms like itching, rhinorrhea, mucus secretion, and smooth muscle contraction in the airways, while AAS results in breathing difficulties, wheezing, chest tightness, and coughing [7]. FAs result from hypersensitivity to specific food allergens and can lead to symptoms such as hypotension, shock, and gastrointestinal reactions [8]. Therefore, new methods are urgently needed to prevent and treat allergic diseases.

Mast cells, present in the skin and mucosal tissues, are the main cells involved in type 1 allergic diseases [9]. IgE-dependent degranulation promotes the production and secretion of mediators such as histamine, cysteinyl leukotrienes (LTC4, LTD4), prostaglandins (PGD2, PGI2), cytokines (e.g., IL-4, IL-13), and chemokines, all of which are major contributors to allergic inflammation [10]. For example, histamine is released in the early stages of the allergic response through degranulation, causing vasodilation and increased vascular permeability [11]. Cytokines, through the activation of mast cells and basophils, amplify the inflammatory response [12]. Cysteinyl leukotrienes promote bronchoconstriction, mucus secretion, and immune cell activation, while prostaglandins contribute to vasodilation. Both of these factors attract neutrophils, stimulating the late-phase allergic response [13,14].

Mast cell degranulation is mediated by the IgE-dependent high-affinity IgE receptor (FcεRI) [15]. When IgE bound to FcεRI of mast cells cross-links with a specific antigen (allergen), it induces the release of various chemical mediators (e.g., histamine, β-hexosaminidase, and prostaglandins) and inflammatory cytokines (e.g., TNF-α and IL-13), leading to the activation and degranulation of mast cells [16,17]. FcεRI is a heterotetrameric receptor composed of a homodimer of an α subunit to which IgE binds and β and γ subunits containing an immunoreceptor tyrosine-based activation motif (ITAM) [15]. In humans, FcεRI exists in both a tetrameric form (αβγ2) and a trimeric form (αγ2), whereas in rodents, only the tetrameric form is present [18]. Upon allergen stimulation, FcεRI aggregation activates Src family kinases, including Lyn, Fyn, and Syk. Following the binding of the antigen-IgE complex to the α subunit, Lyn phosphorylates tyrosine residues within the ITAMs of the β and γ subunits, creating docking sites for Syk [19,20]. Activated Syk subsequently phosphorylates the linker for activation of T cells (LAT), initiating downstream signaling cascades involving phospholipase C (PLC)-γ1 and protein kinase C (PKC) [21,22]. In addition, Fyn phosphorylates LAT and further facilitates signaling through the PI3K-Akt pathway [23,24]. LAT phosphorylation ultimately activates PLC-γ1 and PKC-δ, which, through the mitogen-activated protein kinase (MAPK) pathway (ERK, JNK, p38), promote the release of histamine, cytokines, and leukotrienes, thereby driving mast cell degranulation and allergic responses [25,26].

Currently, allergic disease treatments primarily involve medications such as antihistamines, corticosteroids, and leukotriene antagonists [27]. While these drugs effectively alleviate symptoms, they fail to address the underlying cause—mast cell activation. Moreover, they may cause side effects such as dry eyes, nausea, vomiting, nasal itching, headaches, anxiety, and drowsiness [28]. Given these limitations, there is growing interest in mast cell stabilizers that can inhibit mast cell activation, a key driver of allergic reactions. These stabilizers are classified based on their mechanisms of action, including anti-IgE agents (e.g., omalizumab, quilizumab), Ca²⁺ influx inhibitors (e.g., cromolyn), Syk inhibitors that block intracellular signaling (e.g., piceatannol, curcumin), and PKC family inhibitors (e.g., calphostin C, sphingosine) [29]. As the demand for safer and more effective treatments increased, the development of therapies based on natural compounds has emerged as a promising focus.

Lonicera caerulea, belonging to the Caprifoliaceae family, is widely distributed across Northern Europe, North America, and Northeast Asia. This fruit is oblong, with a deep blue-to-purple color [30]. Lonicera caerulea fruit is a rich source of anthocyanins, especially cyanidin-3-glucoside (C3G), which constitutes over 60% of its total phenolic content. Other significant polyphenols include chlorogenic acid, caffeoylquinic acid, quercetin, and catechin, though in smaller amounts [31,32,33]. Various studies have demonstrated the fruit’s physiological benefits. In a paw edema model using 4-week-old male ICR mice (n = 4) administered with LPS (1 mg/kg), oral administration of Lonicera caerulea fruit extract (300 mg/kg, p.o.) for four days, along with treatment of LPS-stimulated RAW 264.7 cells (300 μg/mL), significantly reduced inflammation and oxidative stress by inhibiting the MAPK/NF-κB pathway and enhancing nuclear factor erythroid-2-related factor 2 (Nrf2) and manganese-dependent superoxide dismutase (MnSOD) expression [34]. In 6-week-old male Sprague-Dawley mice (n = 6) fed a high-fat diet, eight weeks of oral administration of Lonicera caerulea (250 mg/kg, p.o.) improved lipid profiles by activating the Nrf2-ARE pathway [35]. Additionally, 84 days of oral administration of Lonicera caerulea (400 mg/kg, p.o.) in 6-week-old female ICR mice (n = 6) on a high-fructose diet improved obesity and liver health, influencing antioxidant systems (catalase, glutathione), the AMP-activated protein kinase (AMPK) pathway, and glucose metabolism (UCP2, adiponectin) was administered [36]. The primary constituent of Lonicera caerulea fruit, C3G has been shown to exhibit various biological activities. Oral administration of C3G (0.2% wt/wt, p.o.) for six weeks suppressed atherosclerosis in diabetic male apoE−/−mice (6 weeks old, STZ-induced, n = 5–6) by improving endothelial function via AMPK regulation and restoration of eNOS phosphorylation [37]. In an oxidative stress model using HT22 cells (glutamate, 5 mM, 18 h), treatment with C3G (0–100 μM, 24 h) promoted neuronal survival by activating the ERK and Nrf2 pathways, upregulating anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) [38]. Oral administration of C3G (500 mg/kg, p.o.) for eight weeks improved glucose tolerance and hepatic steatosis in a high-fat diet-induced C57BL/6J and PPARα−/−mice (n = 7–10), by activating PPARα, which regulates fatty acid oxidation and ketogenesis [39]. C3G also showed anti-allergic effects; oral administration of C3G (1.2 mg/mouse/day, p.o.) for 25 days alleviated allergic airway inflammation in an ovalbumin (OVA)-induced asthma model (4-week-old male BALB/c mice, 14–16 g, n = 5) by modulating Th2 cytokines and inhibiting the IL-1Rα-STAT6 pathway [40]. Similarly, 12 days of C3G (25 mg/kg, p.o.) in an OVA-induced asthma model (6–8-week-old male BALB/c mice, n = 5) mitigated allergic airway inflammation by modulating histamine, IgA levels, and balancing Th1/Th2 immunity [41]. Additionally, C3G (200 μmol/kg BW, 1 h, p.o.) inhibited passive cutaneous anaphylaxis in IgE-sensitized mice (5-week-old male ICR, 100 ng/ear, n = 4) by suppressing mast cell degranulation from anti-DNP IgE-stimulated RBL-2H3 mast cells elucidated [42]. These results suggest that C3G plays a crucial role in managing allergic reactions. Therefore, the consumption of natural products containing C3G may be beneficial for the prevention and treatment of allergic responses. However, the anti-allergic effects and underlying mechanisms of C3G-rich Lonicera caerulea fruit have not yet been fully elucidated.

This study aims to evaluate the potential of Lonicera caerulea fruit extract (HR2302-30E) as a functional food ingredient with anti-allergic properties. We assessed its bioactive components, including total phenolic and flavonoids and C3G, and evaluated its antioxidant activity. Additionally, the anti-allergic effects of HR2302-30E and C3G were investigated by measuring the secretion of β-hexosaminidase and histamine from IgE-stimulated RBL-2H3 mast cells. Furthermore, we explored the underlying mechanisms by analyzing key signaling pathways, such as FcεRI, Syk, Fyn, Src, PLCγ1, PKCδ, and MAPK. These evaluations provided valuable insights into the effects of mast cell degranulation and the mitigation of allergic reactions.

2. Materials and Methods

2.1. Chemicals

C3G was obtained from Chemface (Wuhan, China). DPPH, ABTS, ascorbic acid, gallic acid, quercetin, Folin–Ciocalteu phenol reagent, potassium ferricyanide, potassium persulfate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), acetic acid, 2,2-azobis(2-amidino propane), 2,4,6-tripyridyl-s-triazine, dihydrochloride, fluorescein sodium salt, sodium nitrite, sodium carbonate, and dimethyl sulfoxide were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Formic acid, acetonitrile, HCl, and methanol were purchased from J. T. Baker (Phillipsburg, NJ, USA). Trypsin-ethylenediaminetetraacetic acid (0.05%), penicillin/streptomycin, phosphate-buffered saline (pH 7.4), minimum essential medium (MEM), and fetal bovine serum were purchased from Gibco (Waltham, MA, USA). MTT assay was performed by DoGenBio (Seoul, Republic of Korea). Dinitrophenyl-human serum albumin (DNP-HSA), anti-DNP-IgE, and 4-nitrophenyl N-acetyl-β-d-glucosaminide, sodium biocarbonate, sodium carbonate, sodium citrate dihydrate, citric acid, and PP2 were purchased from Sigma Aldrich Co.(St. Louis, Mo, USA). All anti-bodies used for western blotting were obtained from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bovine serum albumin and enhanced chemiluminescence (ECL) kits were purchased from Bio-Rad (Hercules, CA, USA).

2.2. Sample Preparation

Lonicera caerulea fruits were purchased from Dong-Yang Herb (Seoul, Republic of Korea) in 2024. Fresh Lonicera caerulea fruits were mixed with 30% ethanol and extracted at 95 °C for 4 h using a reflux apparatus. After filtration with a 106-μm pore size test sieve (Chung Gye Inc., Seoul, Republic of Korea), the extract was concentrated using a vacuum rotary evaporator (Eyela, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) to a soluble solid content of 10 °Bx and powdered. To reduce the stickiness of the extract, a concentrate of Lonicera caerulea fruit rich in sugar and maltodextrin (Samyang Corp., Seoul, Republic of Korea) was mixed in a 1:1 ratio. Lonicera caerulea fruit extract (HR2302-30E) was obtained via lyophilization using a lyophilizer (Labcono, Kansas City, MO, USA). C3G (ChemFace, Wuhan, China) was diluted in dimethyl sulfoxide.

2.3. Total Phenolic and Flavonoid Contents

Total phenolic content was measured using the Folin–Ciocalteu method [43]. Briefly, 510 µL of distilled water and 40 µL of Folin–Ciocalteu reagent were mixed with 200 µL of sample, reacted at 27 °C for 6 min, and mixed with 250 µL of 7% (w/v) sodium carbonate anhydrous solution. After reacting at 27 °C for 90 min, absorbance was measured at 750 nm using a microplate reader (Epoch; BioTek, Winooski, VT, USA). Total phenolic content is expressed as mg gallic acid equivalent (GAE)/g gallic acid.

Total flavonoid content was measured as described by Benítez et al. [44], with slight modifications. To 100 µL of the sample, 400 µL of distilled water and 30 µL of 5% sodium nitrite were added and reacted for 5 min. Then, 30 µL of 10% aluminum chloride was added and reacted at 27 °C for 6 min. The reaction was terminated by adding 200 µL of 1 M sodium hydroxide, and absorbance was measured at 410 nm using a microplate reader. Total flavonoid content is expressed as mg quercetin equivalent (QE)/g quercetin.

2.4. Antioxidant Activity Assays

The DPPH assay was conducted according to Kim et al. [45], with slight modification. Briefly, 0.1 mL of sample and 0.2 mM DPPH solution (0.9 mL) were mixed and reacted for 10 min in the dark at 27 °C. Absorbance was measured at 517 nm using a microplate reader. DPPH radical scavenging activity was calculated using the following equation:

DPPH radical scavenging activity (%) = (1 − [A_sample/A_blank ]) × 100

For the ABTS assay, the procedure by Re et al. [46] was used, with adjustments. A total of 7 mM ABTS and 2.45 mM potassium persulfate were mixed in a 2:1 ratio and reacted for 16 h in the dark at 27 °C. ABTS solution was prepared by diluting the mixture with ethanol until the absorbance reached 0.7 ± 0.02. Then, ABTS+ solution (1 mL) and sample solutions of various concentrations (10 μL) were mixed and reacted for 6 min, and absorbance was measured at 734 nm. ABTS radical scavenging activity was calculated using the following equation:

ABTS radical scavenging activity (%) = (1 − [A_sample/A_blank]) × 100

Next, fluorescence recovery after photobleaching (FRAP) assay was carried out following the procedure described by Benzie and Strain [47], with minor modifications. FRAP solution was prepared by mixing 300 mM acetate buffer (pH 3.6), 2,4,6-tripyridyl-s-triazine solution (10 mL), and 20 mM FeCl₃ solution at a ratio of 10:1:1 (v/v). The sample (100 μL) and FRAP solution (900 μL) were mixed and incubated at 37 °C for 6 min, and absorbance was measured at 593 nm. FRAP is expressed as mM Trolox equivalent (TE)/g Trolox.

The ORAC assay was performed as described by Ou et al. [48], with some modifications. The samples were diluted with 75 mM sodium potassium phosphate buffer, and the diluted sample (25 μL) was mixed with fluorescein (150 μL) and added to a black 96-well plate. Then, 18 mM APPH (25 μL) was transferred to each well and reacted at 37 °C for 15 min. Fluorescence was measured using a fluorometer (SpectraMax Gemini EM; Molecular Devices, Sunnyvale, CA, USA) at excitation and emission wavelengths of 485 and 530 nm, respectively. ORAC value is expressed in mg TE/g Trolox.

2.5. HPLC Analysis of C3G

C3G content was analyzed as described by Ji et al. and Guo et al. [49,50], with slight modifications. HPLC-PDA analysis was performed using the Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with the Dionex Ultimate 3000 photodiode array (PDA-3000; Thermo Fisher Scientific, Waltham, MA, USA). Chromatography was performed on the Kromasil C18 column (4.6 mm × 250 mm, 5.0 μm; Tedia, Rio de Janeiro, Brazil), and the column temperature was maintained at 30 °C. The mobile phases consisted of 0.1% (v/v) formic acid in distilled water (A) and 0.1% (v/v) formic acid in acetonitrile (B). The parameters of HPLC analysis were as follows: 0–20 min: 90% A, 10% B; 20–21 min: 80% A, 20% B; 21–27 min: 20% A, 80% B; 27–28 min: 20% A, 80% B; 28–35 min: 90% A, 10% B gradient system. The analysis time was 35 min, the detection wavelength was 516 nm, the sample injection volume was 10 μL, and the flow rate was 1.0 mL. HR2302-30E sample (100 mg) was mixed with 10 mL of 0.1% (v/v) HCl-methanol and extracted via sonication (POWER SONIC 505; Hwashintech, Daegu, Republic of Korea) for 60 min. The solution was filtered through a 0.45-μm PTFE syringe filter and used as the test solution. A stock solution was also prepared by mixing C3G (200 mg) with 10 mL of 0.1% (v/v) HCl-methanol. The stock solution was diluted with 0.1% (v/v) HCl-methanol to prepare standard solutions with concentrations of 12.5–200 μg/mL.

2.6. Cell Culture

The RBL-2H3 rat basophilic leukemia mast cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in MEM containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂.

2.7. Cell Viability

RBL-2H3 cells were seeded in a 48-well plate at 5 × 10⁴ cells/mL and cultured for 24 h. Then, cells were cultured with various concentrations of HR2302-30E and C3G for 1 h. After treatment, the medium was removed and replaced with 100 μL of MEM containing 10% MTT reagent, and the cells were incubated at 37 °C for 3 h. Cell viability was assessed at 450 nm using a microplate reader (Epoch; Bitek Instruments, Inc., Winooski, VT, USA). The cell viability was calculated using the following equation:

Cell Viability (%) = (A of study group/A of control group) × 100%

2.8. Measurement of β-Hexosaminidase and Histamine Levels

The RBL-2H3 cells were seeded in a 24-well plate at 5 × 10⁵ cells/mL with anti-DNP-IgE (500 ng/mL) and cultured for 24 h. IgE-sensitized cells were pretreated with various concentrations of HR2302-30E and C3G for 30 min and stimulated with or without DNP-HSA (250 ng/mL) for 15 min. To measure β-hexosaminidase released from the cells, the culture supernatant (50 μL) was mixed with 50 μL of substrate buffer (1 mM 4-p-nitrophenyl-N-acetyl-β-D-glucosamidiase in 0.1 M citrate buffer, pH 4.5) and incubated at 37 °C for 2 h. Then, 110 μL of 0.1 M carbonate buffer was added to terminate the reaction, and absorbance was measured at 405 nm. To measure histamine released from the cells, the culture supernatant (100 μL) was mixed with 1 M NaOH (20 μL) and 1% O-phthalaldehyde (25 μL) and reacted for 5 min. After the reaction was terminated by adding 10 μL of 3 M HCl, fluorescence was measured at an excitation wavelength of 360 nm and emission wavelength of 450 nm using a fluorometer (SpectraMax Gemini EM; Molecular Devices, Sunnyvale, CA, USA).

2.9. Western Blotting

The cultured cells were washed with phosphate-buffered saline, lysed by adding the radioimmunoprecipitation assay buffer, and centrifuged. Protein concentration in the supernatant was quantified using a bovine serum albumin protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated via electrophoresis on a 10% sodium dodecyl sulfate-sulfate-polyacrylamide gel. The separated proteins were transferred onto a polyvinylidene fluoride membrane at 100 V for 1 h and blocked with 5% skim milk at room temperature for 1 h. After incubating with primary antibodies diluted 1:1000 to 1:5000 at 4 °C for 16 h, they were incubated with horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG diluted 1:3000 at room temperature for 1 h. Protein expression was visualized using the ChemiDoc MP imaging system (Bio-Rad) after incubating the membrane for photoexposure using an ECL kit. The expression levels were quantified using an imaging densitometer (ImageJ 64-bit Java 1.8.0_112).

2.10. Statistical Analyses

Data are represented as the mean ± standard error of the mean. Statistical analysis was performed using SPSS software (ver. 24.0; IBM Corp., Armonk, NY, USA). The significance of the values was analyzed using one-way analysis of variance (ANOVA), followed by additional analysis with Duncan’s multiple range test and Dunnett’s multiple comparison test. Statistical significance was set at p < 0.05, p < 0.01, and p < 0.001.

3. Results

3.1. Total Phenolic and Flavonoid Contents of HR2302-30E

Various berries, including Lonicera caerulea fruits, are known to have high phenolic and flavonoid contents [51]. Previous studies have shown that polyphenols and flavonoids regulate FcεRI signaling in IgE-induced mast cells, inhibiting mast cell activation and degranulation, which helps prevent allergic reactions [52,53,54]. The total phenolic and flavonoid contents of HR2302-30E determined in this study are presented in Table 1. The total phenolic content of HR2302-30E was 18.73 ± 0.74 mg/g, and the total flavonoid content was 11.83 ± 0.81 mg/g.

3.2. Analysis of HR2302-30E Antioxidant Activity Using Various Chemical Assays

Reactive oxygen species induce inflammation by mobilizing and activating oxidative stress factors and various inflammatory cells. Many inflammatory molecules mediate the diseases caused by allergic reactions [55,56]. Considering the relationship between allergic inflammation and ROS, evaluating the antioxidant potential of HR2302-30E will provide insight into its potential contribution to the modulation of allergic responses. The effects of various processes are evaluated to determine the overall antioxidant capacity [57]. Here, the antioxidant activity of HR2302-30E was measured using four in vitro assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and ORAC assays. The results of the antioxidant analysis are shown in Figure 1 and Table 2. DPPH radical scavenging activity of HR2302-30E was approximately 23.13–65.33% at 1–5 mg/mL, and the half-maximal inhibitory concentration (IC50) was 3.4 mg/mL (Figure 1a). Its ABTS radical scavenging activity was 10.28–43.25%, and its IC50 value was 5.56 mg/mL (Figure 1b). FRAP activity was 0.53–2.63 and 93.84 mmol TE/g (Figure 1c). ORAC value was 181.59 μmol TE/g (Figure 1d).

3.3. Analysis of C3G Content of HR2302-30E

C3G is a major compound of Lonicera caerulea fruit [58]. The C3G content of HR2302-30E was analyzed via high-performance liquid chromatography (HPLC). Figure 2 shows the chromatograms and photodiode array (PDA) spectra of C3G (Figure 2a,c) and HR2302-30E (Figure 2b,c). C3G and HR2302-30E exhibited maximum absorption at 516 nm in the UV wavelength range of 190–600 nm. C3G was separated from HR2302-30E without interference from other substances and showed the same peak retention time. C3G content in HR2302-30E was 7.02 ± 0.17 mg/g.

3.4. Effect of HR2302-30E and C3G on RBL-2H3 Cell Viability

An MTT assay was performed to evaluate the effect of HR2302-30E on the viability of RBL-2H3 cells. As shown in Figure 3, the viability of RBL-2H3 mast cells treated with various concentrations of HR2302-30E and C3G was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, both HR2302-30E (500, 1000, and 2000 μg/mL) and C3G (5 and 10 μg/mL) did not significantly affect the cell viability compared to the negative control; thus, the extract is not cytotoxic to the selected cell line.

3.5. Effects of HR2302-30E and C3G on Anti-DNP-IgE-Induced Degranulation of RBL-2H3 Cells

To evaluate the effect of HR2302-30E and C3G on the degranulation of RBL-2H3 cells, the release of β-hexosaminidase and histamine was measured. In activated mast cells, pre-formed β-hexosaminidase and histamine are released into the granules during degranulation [59]. β-hexosaminidase and histamine levels are widely used as markers of mast cell degranulation, which plays an important role in allergic responses [60,61]. To evaluate the inhibitory effect on degranulation, an early marker of allergic response, IgE-primed RBL-2H3 cells were stimulated with 250 ng/mL DNP-HSA and treated with HR2302-30E (1000 and 2000 μg/mL) and C3G (5 and 10 μg/mL). The release of β-hexosaminidase and histamine was measured to assess the degranulation of RBL-2H3 mast cells. As shown in Figure 4, HR2302-30E and C3G significantly decreased β-hexosaminidase (Figure 4a) and histamine release (Figure 4b) in activated mast cells. PP2, an Src family kinase inhibitor, was used as a positive control. These results suggest that HR2302-30E and C3G inhibit the activation-induced degranulation of mast cells.

3.6. Effects of HR2302-30E and C3G on the FcεRI Signaling Pathway

When allergens bind to IgE bound to FcεRI, FcεRI aggregates, activating the mast cells. Aggregation of FcεRIβ and FcεRIγ induces the activation of Src family tyrosine kinases, Lyn and Fyn, which phosphorylate the tyrosine residues in ITAM, leading to Syk activation. This further causes PLCγ and PKCδ activation and intracellular calcium release, contributing to the activation of the MAPK pathway. PLCγ and PKCδ stimulate the secretion of β-hexosaminidase and histamine, and MAPK leads to the synthesis of inflammatory cytokines [21,62,63]. The MAPK family, including ERK, p38, and JNK, is activated by various stimuli that induce inflammatory responses. These MAPKs are closely associated with degranulation and the secretion of inflammatory cytokines in mast cells [64]. Notably, ERK is activated by IgE receptor stimulation and plays a critical role in mast cell differentiation and proliferation. p38 stimulates mast cell migration and the secretion of IL-4, while JNK plays a role in the release of inflammatory cytokines, including IL-6 and IFN-γ [65]. Therefore, we assessed the effects of HR2302-30E and C3G on the activation of the FcεRI pathway using Western blotting. HR2302-30E and C3G significantly reduced the expression levels of the FcεRI subunits FcεRIβ and FcεRIγ (Figure 5a,b) and inhibited the phosphorylation of Syk, Fyn, and Src in a dose-dependent manner (Figure 5c,d). Furthermore, HR2302-30E and C3G decreased the phosphorylation of key components of the MAPK pathway, including p38, ERK, and JNK (Figure 6a,b), and significantly reduced the phosphorylation of PLCγ and PKCδ, which are involved in mast cell degranulation (Figure 6c,d). These results suggest that HR2302-30E and C3G contribute to the inhibition of mast cell degranulation by suppressing FcεRI receptor activation and blocking the initial signaling pathways involving Syk, Fyn, and Src. Additionally, HR2302-30E and C3G may exhibit anti-inflammatory effects by inhibiting the secretion of cytokines that trigger inflammatory responses and the release of β-hexosaminidase and histamine during the mast cell degranulation process. The anti-allergic properties of HR2302-30E and C3G were confirmed through their inhibition of the FcεRI signaling pathway and the mechanisms underlying the production of inflammatory cytokines and histamine (Figure 7).

4. Discussion

The prevalence of allergic diseases and conditions, including allergic rhinitis, atopic dermatitis, and asthma, is increasing worldwide, posing a significant economic burden on affected individuals [66,67]. Several polyphenols are known to possess anti-allergic activities by inhibiting mast cell activation in vitro. For example, tricin (100–500 μg/mL) inhibits the phosphorylation of protein kinase B (Akt), p38, ERK, and JNK in activated RBL-2H3 mast cells, as well as Syk, Lyn, PLCγ, and PKCδ [54]. Similarly, saponarin (80 μM) inhibits the expression of FcεRIα/γ in IgE-activated RBL-2H3 mast cells and suppresses mast cell activation by reducing the phosphorylation of Syk, PLCγ, ERK, JNK, and p38 [55]. Quercetin inhibits mast cell activation by reducing the surface expression of FcεRI and cytokine production (IL-6, IL-33) in IgE-activated BMMC (bone marrow-derived mast cells) [53].

Lonicera caerulea fruit is a rich source of C3G. C3G has been shown to exert anti-allergic effects through regulation of IL-1Rα-STAT6 signaling, strengthening of the intestinal epithelial barrier, and regulation of Th1/Th2 immune balance in the OVA-induced asthma mice model [40,41]. Additionally, anti-DNP inhibited β-hexosaminidase and histamine secretion from IgE-stimulated RBL-2H3 mast cells and suppressed passive cutaneous anaphylaxis in IgE-sensitized mice [42]. Despite these anti-allergic properties of C3G, studies specifically focusing on the anti-allergic effects of Lonicera caerulea fruit have not yet been conducted. In this study, we evaluated the potential anti-allergic effects of HR2303-30E and C3G. We assessed its impact on the secretion of β-hexosaminidase and histamine in RBL-2H3 mast cells. Additionally, we investigated the modulation of key signaling pathways, including FcεRI, Syk, Fyn, Src, PLCγ1, PKCδ, and MAPK.

Lonicera caerulea fruit has various components, such as ascorbic acid, anthocyanins, flavonoids, and phenolic acids, and exhibits antioxidant activity [68,69]. We measured the total phenolic content, total flavonoid content, and antioxidant activity of HR2302-30E in this study. First, we measured the phenolic content of Lonicera caerulea berries. Total phenolic and flavonoid contents of HR2302-30E were 18.73 ± 0.74 mg GAE/g and 11.83 ± 0.81 mg QE/g, respectively, which are higher than those reported in previous studies (4.22–11.11 mg GAE/g and 4.36–11.37 mg QE/g, respectively) [70,71,72]. Antioxidant activity cannot be tested using a single method and must be analyzed using various procedures [57]. Here, the antioxidant activity of HR2302-30E was measured using four methods: DPPH, ABTS, FRAP, and ORAC assays. DPPH radical scavenging assay is based on the electron donation of antioxidants to neutralize DPPH, a stable chromogenic radical with a deep purple color [73]. The ABTS test is based on a decolorization reaction, in which a stable radical cation (ABTS ^•+), a blue-green-emitting group, is removed by antioxidants either through direct reduction via electron donation or radical quenching via hydrogen atom donation [74]. The FRAP method measures the potential of a reducing agent (antioxidant) to reduce ferric ion (Fe³⁺)-ligand to blue ferrous complex (Fe²⁺) [75]. The ORAC assay measures the radical chain scavenging activity of antioxidants by monitoring the inhibition of peroxyl radical oxidation [76]. DPPH radical scavenging activity (IC50), ABTS radical scavenging activity (IC50), FRAP activity, and ORAC value of HR2302-30E were 3.40 mg/mL, 5.56 mg/mL, 93.84 mmol TE/g, and 181.59 μmol TE/g, respectively. In a previous study, the antioxidant activity of Lonicera caerulea fruit varied from 5.83 to 7.8 mg/mL in the DPPH assay, from 27.96 to 46.9 μM TE/g in the FRAP assay, and from 267.19 to 262.44 μM TE/g in the ORAC assay [70]. These differences in results may be due to the different cultivars, cultivation locations, and harvest periods.

The major representative compound of Lonicera caerulea fruit is C3G of the anthocyanin series [77]. Therefore, we measured the content of C3G, a representative anthocyanin, in HR2302-30E. C3G content of HR2302-30E was found to be 7.02 ± 0.17 mg/g. C3G accounts for 70–90% of the anthocyanin profile and is present at 0.68~41.16 mg/mL in Lonicera caerulea fruit [51,59,70,78,79,80].

Allergy is the overreaction of the immune system to a generally harmless substance, accompanied by an inflammatory response. Mast cell activation is the response to the cross-linking of allergens to the IgE/FcεRI complex, which induces allergic immune responses. FcεRI receptor consists of an α-subunit to which IgE binds and β- and γ-subunits involved in intracellular signaling. Upon receptor aggregation, initial phosphorylation of ITAM in the β- and γ-subunits is caused by Lyn and Fyn, respectively, which are Src family kinases. Activated ITAM serves as a docking site for the activation of Syk [81,82,83]. Incubation of RBL-2H3 cells with IgE in vitro increases the expression of FcεRI on the cell surface [84,85]. Moreover, FcεRI aggregate formation is activated by Src family kinases, such as Lyn, Fyn, and Syk [82]. HR2302-30E and C3G significantly decreased the expression of FcεRIβ and FcεRIγ and inhibited the phosphorylation of Src, Fyn, and Syk in anti-DNP-IgE-activated RBL-2H3 mast cells. These results indicate that HR2302-30E and C3G inhibit mast cell activation by blocking the binding sites of FcεRI and interfering with the signaling pathways of Src, Fyn, and Syk.

Tyrosine-phosphorylated Syk induces the activation of downstream signaling proteins, such as LAT, PLCγ, and PKCδ. Phosphorylated LAT provides a binding site for PLCγ, which regulates the elevation of intracellular Ca²⁺ levels and activation of PKCδ [86,87]. Phosphorylation of PLCγ and PKCδ stimulates the secretion of histamine, β-hexosaminidase, and inflammatory cytokines, which induce allergic responses [88]. Activation of Fyn and Syk induces MAPK. MAPK activation plays a key role in the production of inflammatory mediators, including cytokines, and recruitment of immune cells [89]. MAPK activation of ERK regulates the proliferation, migration, and differentiation of mast cells under allergic conditions, as well as the activation of JNK and p38, which produces inflammatory cytokines and mediators in abnormally activated mast cells [65]. It is the major mechanism regulating allergic inflammation. In the present work, HR2302-30E and C3G inhibited the phosphorylation of PLCγ and PKCδ. Additionally, HR2302-30E and C3G significantly suppressed the phosphorylation of p38, ERK, and JNK in mast cells with abnormally activated MAPK signaling pathways. These findings suggest that HR2302-30E and C3G may inhibit the release of allergy mediators, such as Ca²⁺, histamine, and β-hexosaminidase by blocking the phosphorylation of PLCγ and PKCδ. Furthermore, by suppressing the MAPK signaling pathway, they may block the inflammatory response, thereby contributing to their anti-allergic and anti-inflammatory effects. Our study demonstrated the antiallergic effects of HR2302-30E and C3G, particularly through the regulation of various signaling pathways (FcεRI, Src, Fyn, Syk, PLCγ, PKCδ, MAPK), which modulate the release of β-hexosaminidase and histamine in RBL-2H3 mast cells. These results suggest the potential of HR2302-30E and C3G as natural products for regulating mast cell activation in allergy treatments.

Increased intracellular calcium levels play a key role in stimulating mast cell activation and extracellular secretion of granules [90]. Activated mast cells release allergic mediators, such as histamine, β-hexosaminidase, serotonin, and protease, via immediate degranulation and induce allergic inflammation via cytokine gene expression [91]. Histamine induces inflammatory responses via vasodilation, increased vascular permeability, and edema; β-hexosaminidase induces allergic inflammation [11]. Here, HR2302-30E and C3G significantly inhibited the release of histamine and β-hexosaminidase from mast cells in a concentration-dependent manner. These results imply that HR2302-30E and C3G inhibit mast cell activation and degranulation. Since the inhibition of mast cell activation and degranulation is regarded as a key target in anti-allergic mechanisms, HR2302 has been confirmed to have the potential to be used as a functional food ingredient with anti-allergic properties.

In this study, we evaluated the anti-allergic activity of HR2302-30E using the RBL-2H3 cell line. The RBL-2H3 cell line, derived from rat basophils, exhibits responses similar to those of mast cells and basophils [92], such as the release of β-hexosaminidase and histamine, as well as the expression of FcεRI [93]. Consequently, it is widely used to investigate IgE receptor-mediated degranulation reactions [54,94]. However, the anti-allergic effects of HR2302-30E and C3G could not be completely verified in this in vitro study. First, since only the RBL-2H3 cell line was used, it was challenging to assess the reproducibility of the results in other cell types. Second, the use of a cell line, rather than primary cells, may not accurately reflect the in vivo environment. Third, the use of rat cells rather than human cells may limit the extent to which these results reflect human allergic responses. Notably, the expression of FcεRI differs between humans and rodents. In humans, FcεRI exists in both the tetrameric (αβγ₂) and trimeric (αγ₂) forms, whereas in rodents, only the tetrameric form is present. These structural differences may result in rodents not displaying the same level of IgE-mediated hypersensitivity as humans, potentially leading to differences in how FcεRI mediates immune responses. Therefore, the results obtained from rodent models may have limitations in interpreting human allergic diseases [95]. To address these limitations, future studies should verify the results using primary human basophils or mast cells and evaluate the reproducibility of the findings using various cell lines. Additionally, in vivo studies using animal models of atopic dermatitis or allergic rhinitis are needed to validate the findings in a biological context. Recent research has emphasized that the effects of foods beneficial for allergies may be mediated through epigenetic mechanisms, such as DNA methylation and histone modification [18,96]. Future research should also evaluate the impact of these compounds on epigenetic pathways.

5. Conclusions

In summary, this study demonstrated that HR2302-30E and C3G exert anti-allergic effects by inhibiting the activation of mast cells stimulated by IgE. HR2302-30E exhibited strong antioxidant activity based on its high phenolic and flavonoid contents. In addition, the content of cyainidin-3-glucoside (C3G), a representative anthocyanin compound of HR2302-30E, was analyzed to be 7.02 ± 0.17 mg/g. HR2302-30E and C3G were shown to inhibit the release of β-hexosaminidase and histamine in RBL-2H3 cells. The anti-allergic mechanisms of HR2302-30E and C3G are associated with the suppression of FcεRI and Fyn/Syk pathway activation in activated mast cells, as well as the inhibition of downstream signaling molecules, such as PLCγ, PKCδ, and MAPK. This ultimately regulates mast cell degranulation and allergic inflammation. In this study, the antiallergic effects of HR2302-30E and C3G were evaluated in vitro using mast cells. However, future research should validate these findings by using primary human mast cells or basophils and assess reproducibility across various cell lines. Additionally, further in vivo studies utilizing animal models of allergic diseases, such as atopic dermatitis or allergic rhinitis, are essential to confirm the observed effects. Moreover, exploring the potential impact of HR2302-30E and C3G on epigenetic regulation in allergic inflammation could provide valuable insights into their therapeutic potential. Taken together, our results indicate that HR2302-30E and C3G have the potential as natural functional foods for modulating mast cell-mediated allergic diseases.

Author Contributions

Conceptualization, Y.-E.C. and S.-H.P.; methodology, Y.-E.C., J.-M.Y. and H.-W.Y.; software, C.-W.J., H.-W.Y. and S.-H.P.; validation, J.-M.Y. and C.-W.J.; formal analysis, J.-M.Y., C.-W.J. and H.-W.Y.; investigation, Y.-E.C. and S.-H.P.; resources, H.-D.J. and J.-H.C.; data curation, Y.-E.C. and C.-W.J.; writing—original draft preparation, Y.-E.C. and S.-H.P.; writing—review and editing, Y.-E.C. and J.-H.C.; visualization, S.-H.P.; supervision, H.-D.J. and J.-H.C.; project administration, H.-D.J. and J.-H.C.; funding acquisition, H.-D.J. and J.-H.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. Antioxidant activity of HR2302-30E. (a) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. (b) 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. (c) Ferric reducing antioxidant power (FRAP). (d) Oxygen radical absorbance capacity (ORAC) plot. ORAC values are expressed as the net area under the curve. Data are represented as the mean ± standard error of the mean (SEM) of three independent experiments. Different letters on the bars indicate significant differences (p [less than] 0.05) in the Duncan multiple range test. a~g, significant differences among various samples. AsA, Ascorbic acid.

Figure 2. High-performance liquid chromatography (HPLC) chromatograms and photodiode array (PDA) spectra of cyanidin-3-glucoside (C3G) (a,c) and HR2302-30E (b,d).

Figure 3. Effects of HR2302-30E and C3G on the viability of RBL-2H3 mast cells. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Negative control: untreated cells. Data are represented as the mean ± SEM of three independent experiments. The statistical analyses were performed using the Dunnett t-test. PP2 is a general Src family kinase inhibitor. −, No treatment.

Figure 4. Effects of HR2302-30E and C3G on (a) β-hexosaminidase and (b) histamine release in RBL-2H3 mast cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (500, 1000, and 2000 μg/mL) and C3G (5 and 10 μg/mL). Negative control: non-IgE-primed RBL-2H3 cells without DNP-HSA; positive control: IgE-primed RBL-2H3 cells with DNP-HSA and PP2. Data are represented as the mean ± SEM of the three independent experiments. The statistical analyses were performed using the Dunnett t-test. PP2 is a general Src family kinase inhibitor. * p [less than] 0.05, ** p [less than] 0.01 and *** p [less than] 0.001 vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); ### p [less than] 0.001 vs. control. −, No treatment, +, Treatment.

Figure 5. Effects of HR2302-30E and C3G on the high-affinity IgE receptor (FcεRI) signaling cascade in RBL-2H3 cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (1000 and 2000 μg/mL) and C3G (5 and 10 μg/mL). The protein expression of (a,b) FcεRI β and FcεRI γ, (c,d) Syk, Fyn, and Src were determined using immunoblotting. Data are represented as the mean ± SEM of the three independent experiments. The statistical analyses were performed using the Dunnett t-test. PP2 is a general Src family kinase inhibitor. * p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001 vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); ### p [less than] 0.001 vs. control. −, No treatment, +, Treatment.

Figure 5. Effects of HR2302-30E and C3G on the high-affinity IgE receptor (FcεRI) signaling cascade in RBL-2H3 cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (1000 and 2000 μg/mL) and C3G (5 and 10 μg/mL). The protein expression of (a,b) FcεRI β and FcεRI γ, (c,d) Syk, Fyn, and Src were determined using immunoblotting. Data are represented as the mean ± SEM of the three independent experiments. The statistical analyses were performed using the Dunnett t-test. PP2 is a general Src family kinase inhibitor. * p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001 vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); ### p [less than] 0.001 vs. control. −, No treatment, +, Treatment.

Figure 6. Effects of HR2302-30E and C3G on mitogen-activated protein kinase (MAPK), phospholipase C (PLC)-γ, and protein kinase C (PKC)-δ levels in RBL-2H3 cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (1000 and 2000 μg/mL) and C3G (5 and 10 μg/mL). The protein expression of (a,b) p38, ERK, and JNK, (c,d) PLCγ and PKCδ were determined using immunoblotting. Data are represented as the mean ± SEM of the three independent experiments. The statistical analyses were performed using the Dunnett t-test. PP2 is a general Src family kinase inhibitor. * p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001 vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); ### p [less than] 0.001 vs. control. −, No treatment, +, Treatment.

Figure 6. Effects of HR2302-30E and C3G on mitogen-activated protein kinase (MAPK), phospholipase C (PLC)-γ, and protein kinase C (PKC)-δ levels in RBL-2H3 cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (1000 and 2000 μg/mL) and C3G (5 and 10 μg/mL). The protein expression of (a,b) p38, ERK, and JNK, (c,d) PLCγ and PKCδ were determined using immunoblotting. Data are represented as the mean ± SEM of the three independent experiments. The statistical analyses were performed using the Dunnett t-test. PP2 is a general Src family kinase inhibitor. * p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001 vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); ### p [less than] 0.001 vs. control. −, No treatment, +, Treatment.

Figure 7. HR2302-30E and C3G suppress mast cell degranulation by inhibiting FcεRI signaling pathway. HR2302-30E and C3G inhibit FcεRI signaling by suppressing Src family kinases (Syk, Fyn) and downstream pathways (PLCγ, PKCδ, MAPK), thereby reducing the release of allergic mediators.

Table 1

Total phenolic and total flavonoid contents of HR2302-30E.

Contents	HR2302-30E
Total phenolic contents (mg GAE 1/g)	18.73 ± 0.74
Total flavonoid contents (mg QE 2/g)	11.83 ± 0.81

¹ Gallic acid equivalent, ² Quercetin equivalent.

Table 2

Antioxidant activity of HR2302-30E.

Contents	HR2302-30E
DPPH (mg/mL, IC50)	3.40 ± 0.22
ABTS (mg/mL, IC50)	5.56 ± 0.12
FRAP (mmol TE 1/g)	93.84 ± 1.04
ORAC value (μmol TE 1/g)	181.59 ± 6.88

¹ Trolox equivalent.

References

1. Hadi, H.A.; Tarmizi, A.I.; Khalid, K.A.; Gajdács, M.; Aslam, A.; Jamshed, S. The epidemiology and global burden of atopic dermatitis: A narrative review. Life; 2021; 11, 936. [DOI: https://dx.doi.org/10.3390/life11090936]

2. Bylund, S.; von Kobyletzki, L.B.; Svalstedt, M.; Svensson, Å. Prevalence and incidence of atopic dermatitis: A systematic review. Acta Derm. Venereol.; 2020; 100, pp. 320-329. [DOI: https://dx.doi.org/10.2340/00015555-3510] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32412646]

3. Ito, Y.; Kato, T.; Yoshida, K.; Takahashi, K.; Fukutomi, Y.; Nagao, M.; Adachi, Y. Prevalence of allergic diseases across all ages in Japan: A nationwide cross-sectional study employing designated allergic disease medical hospital network. JMA J.; 2023; 6, pp. 165-174.

4. Ha, J.; Lee, S.W.; Yon, D.K. Ten-year trends and prevalence of asthma, allergic rhinitis, and atopic dermatitis among the Korean population, 2008–2017. Clin. Exp. Pediatr.; 2020; 63, 278. [DOI: https://dx.doi.org/10.3345/cep.2019.01291] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32023407]

5. Wang, J.; Zhou, Y.; Zhang, H.; Hu, L.; Liu, J.; Wang, L.; Wang, T.; Zhang, H.; Cong, L.; Wang, Q. Pathogenesis of allergic diseases and implications for therapeutic interventions. Signal Transduct. Target. Ther.; 2023; 8, 138. [DOI: https://dx.doi.org/10.1038/s41392-023-01344-4] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36964157]

6. Wilson, S.R.; Batia, L.M.; Beattie, K.; Katibah, G.E.; McClain, S.P.; Pellegrino, M.; Estandian, D.M.; Bautista, D.M. The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell; 2013; 155, pp. 285-295. [DOI: https://dx.doi.org/10.1016/j.cell.2013.08.057] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24094650]

7. Dykewicz, M.S.; Hamilos, D.L. Rhinitis and sinusitis. J. Allergy Clin. Immunol.; 2010; 125, pp. S103-S115. [DOI: https://dx.doi.org/10.1016/j.jaci.2009.12.989] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20176255]

8. Deschildre, A.; Lejeune, S. How to cope with food allergy symptoms?. Curr. Opin. Allergy Clin. Immunol.; 2018; 18, pp. 234-242. [DOI: https://dx.doi.org/10.1097/ACI.0000000000000447]

9. Akdis, C.A.; Akdis, M. Mechanisms of allergen-specific immunotherapy and immune tolerance to allergens. World Allergy Organ. J.; 2015; 8, pp. 1-12. [DOI: https://dx.doi.org/10.1186/s40413-015-0063-2] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26023323]

10. Beghdadi, W.; Madjene, L.C.; Benhamou, M.; Charles, N.; Gautier, G.; Launay, P.; Blank, U. Mast cells as cellular sensors in inflammation and immunity. Front. Immunol.; 2011; 2, 37. [DOI: https://dx.doi.org/10.3389/fimmu.2011.00037]

11. Thangam, E.B.; Jemima, E.A.; Singh, H.; Baig, M.S.; Khan, M.; Mathias, C.B.; Saluja, R. The role of histamine and histamine receptors in mast cell-mediated allergy and inflammation: The hunt for new therapeutic targets. Front. Immunol.; 2018; 9, 1873. [DOI: https://dx.doi.org/10.3389/fimmu.2018.01873] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30150993]

12. Mukai, K.; Tsai, M.; Saito, H.; Galli, S.J. Mast cells as sources of cytokines, chemokines, and growth factors. Immunol. Rev.; 2018; 282, pp. 121-150. [DOI: https://dx.doi.org/10.1111/imr.12634]

13. Liu, M.; Yokomizo, T. The role of leukotrienes in allergic diseases. Allergol. Int.; 2015; 64, pp. 17-26. [DOI: https://dx.doi.org/10.1016/j.alit.2014.09.001]

14. Bisgaard, H. Leukotrienes and prostaglandins in asthma. Allergy; 1984; 39, pp. 413-420. [DOI: https://dx.doi.org/10.1111/j.1398-9995.1984.tb01963.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/6208802]

15. Kraft, S.; Kinet, J.P. New developments in FcεRI regulation, function and inhibition. Nat. Rev. Immunol.; 2007; 7, pp. 365-378. [DOI: https://dx.doi.org/10.1038/nri2072] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17438574]

16. Stone, K.D.; Prussin, C.; Metcalfe, D.D. IgE, mast cells, basophils, and eosinophils. J. Allergy Clin. Immunol.; 2010; 125, pp. S73-S80. [DOI: https://dx.doi.org/10.1016/j.jaci.2009.11.017]

17. Ohkawara, Y.; Yamauchi, K.; Tanno, Y.; Tamura, G.; Ohtani, H.; Nagura, H.; Takishima, T. Human lung mast cells and pulmonary macrophages produce tumor necrosis factor-a in sensitized lung tissue after 19B receptor triggering. Am. J. Respir. Cell Mol. Biol.; 1992; 7, pp. 385-392. [DOI: https://dx.doi.org/10.1165/ajrcmb/7.4.385]

18. Potaczek, D.P.; Kabesch, M. Current concepts of IgE regulation and impact of genetic determinants. Clin. Exp. Allergy; 2012; 42, pp. 852-871. [DOI: https://dx.doi.org/10.1111/j.1365-2222.2011.03953.x]

19. Ingley, E. Functions of the Lyn tyrosine kinase in health and disease. Cell Commun. Signal.; 2012; 10, 21. [DOI: https://dx.doi.org/10.1186/1478-811X-10-21] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22805580]

20. Hong, H.; Kitaura, J.; Xiao, W.; Horejsi, V.; Ra, C.; Lowell, C.A.; Kawakami, T. The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn. Blood J. Am. Soc. Hematol.; 2007; 110, pp. 2511-2519. [DOI: https://dx.doi.org/10.1182/blood-2007-01-066092] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17513616]

21. Siraganian, R.P.; de Castro, R.O.; Barbu, E.A.; Zhang, J. Mast cell signaling: The role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants. FEBS Lett.; 2010; 584, pp. 4933-4940. [DOI: https://dx.doi.org/10.1016/j.febslet.2010.08.006] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20696166]

22. Gu, H.; Saito, K.; Klaman, L.D.; Shen, J.; Fleming, T.; Wang, Y.; Pratt, J.C.; Lin, G.; Lim, B.; Kinet, J.-P. et al. Essential role for Gab2 in the allergic response. Nature; 2001; 412, pp. 186-190. [DOI: https://dx.doi.org/10.1038/35084076] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11449275]

23. Nishida, K.; Yamasaki, S.; Hasegawa, A.; Iwamatsu, A.; Koseki, H.; Hirano, T. Gab2, via PI-3K, regulates ARF1 in FcεRI-mediated granule translocation and mast cell degranulation. J. Immunol.; 2011; 187, pp. 932-941. [DOI: https://dx.doi.org/10.4049/jimmunol.1100360] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21653832]

24. Sugie, K.; Jeon, M.S.; Grey, H.M. Activation of naive CD4 T cells by anti-CD3 reveals an important role for Fyn in Lck-mediated signaling. Proc. Natl. Acad. Sci. USA; 2004; 101, pp. 14859-14864. [DOI: https://dx.doi.org/10.1073/pnas.0406168101]

25. Chuck, M.I.; Zhu, M.; Shen, S.; Zhang, W. The role of the LAT–PLC-γ1 interaction in T regulatory cell function. J. Immunol.; 2010; 184, pp. 2476-2486. [DOI: https://dx.doi.org/10.4049/jimmunol.0902876] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20130215]

26. Teegala, L.R.; Elshoweikh, Y.; Gudneppanavar, R.; Thodeti, S.; Pokhrel, S.; Southard, E.; Paruchuri, S.; Southard, E.; Thodeti, C.k.; Paruchuri, S. Protein Kinase C α and β compensate for each other to promote stem cell factor-mediated KIT phosphorylation, mast cell viability and proliferation. FASEB J.; 2022; 36, e22273. [DOI: https://dx.doi.org/10.1096/fj.202101838RRR] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35349200]

27. Burchett, J.R.; Dailey, J.M.; Kee, S.A.; Pryor, D.T.; Kotha, A.; Kankaria, R.A.; Straus, D.B.; Ryan, J.J. Targeting mast cells in allergic disease: Current therapies and drug repurposing. Cells; 2022; 11, 3031. [DOI: https://dx.doi.org/10.3390/cells11193031]

28. Malone, M.; Kennedy, T.M. Review: Side effects of some commonly used allergy medications (decongestants, anti-leukotriene agents, antihistamines, steroids, and zinc) and their safety in pregnancy. Int. J. Aller. Medicat.; 2017; 3, pp. 024-029. [DOI: https://dx.doi.org/10.23937/2572-3308.1510024]

29. Cao, M.; Gao, Y. Mast cell stabilizers: From pathogenic roles to targeting therapies. Front. Immunol.; 2024; 15, 1418897. [DOI: https://dx.doi.org/10.3389/fimmu.2024.1418897] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/39148726]

30. Plekhanova, M.N. Blue honeysuckle (Lonicera caerulea L.)—A new commercial berry crop for temperate climate: Genetic resources and breeding. Eucarpia Symp. Fruit Breed. Genet.; 2000; 538, pp. 159-164. [DOI: https://dx.doi.org/10.17660/ActaHortic.2000.538.25]

31. Sharma, A.; Lee, H.J. Lonicera caerulea: An updated account of its phytoconstituents and health-promoting activities. Trends Food Sci. Technol.; 2021; 107, pp. 130-149. [DOI: https://dx.doi.org/10.1016/j.tifs.2020.08.013]

32. Oszmiański, J.; Wojdyło, A.; Lachowicz, S. Effect of dried powder preparation process on polyphenolic content and antioxidant activity of blue honeysuckle berries (Lonicera caerulea L. var. kamtschatica). LWT Food Sci. Technol.; 2016; 67, pp. 214-222. [DOI: https://dx.doi.org/10.1016/j.lwt.2015.11.051]

33. Kucharska, A.Z.; Sokół-Łętowska, A.; Oszmiański, J.; Piórecki, N.; Fecka, I. Iridoids, phenolic compounds and antioxidant activity of edible honeysuckle berries (Lonicera caerulea var. kamtschatica Sevast). Molecules; 2017; 22, 405. [DOI: https://dx.doi.org/10.3390/molecules22030405] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28273885]

34. Wu, S.; Yano, S.; Chen, J.; Hisanaga, A.; Sakao, K.; He, X.; Hou, D.X. Polyphenols from Lonicera caerulea L. berry inhibit LPS-induced inflammation through dual modulation of inflammatory and antioxidant mediators. J. Agric. Food Chem.; 2017; 65, pp. 5133-5141. [DOI: https://dx.doi.org/10.1021/acs.jafc.7b01599] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28573848]

35. Wang, Y.; Gao, N.; Nieto-Veloza, A.; Zhou, L.; Sun, X.; Si, X.; Tian, J.; Lin, Y.; Jiao, X.; Li, B. Lonicera caerulea polyphenols inhibit fat absorption by regulating Nrf2-ARE pathway mediated epithelial barrier dysfunction and special microbiota. Food Sci. Hum. Wellness; 2023; 12, pp. 1309-1322. [DOI: https://dx.doi.org/10.1016/j.fshw.2022.10.013]

36. Kim, J.W.; Lee, Y.S.; Seol, D.J.; Cho, I.J.; Ku, S.K.; Choi, J.S.; Lee, H.J. Anti-obesity and fatty liver-preventing activities of Lonicera caerulea in high-fat diet-fed mice. Int. J. Mol. Med.; 2018; 42, pp. 3047-3064. [DOI: https://dx.doi.org/10.3892/ijmm.2018.3879] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30221679]

37. Zhang, Y.; Wang, X.; Wang, Y.; Liu, Y.; Xia, M. Supplementation of cyanidin-3-O-β-glucoside promotes endothelial repair and prevents enhanced atherogenesis in diabetic apolipoprotein E–deficient mice. J. Nutr.; 2013; 143, pp. 1248-1253. [DOI: https://dx.doi.org/10.3945/jn.113.177451] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23761653]

38. Sukprasansap, M.; Chanvorachote, P.; Tencomnao, T. Cyanidin-3-glucoside activates Nrf2-antioxidant response element and protects against glutamate-induced oxidative and endoplasmic reticulum stress in HT22 hippocampal neuronal cells. BMC Complement. Med. Ther.; 2020; 20, pp. 213-219. [DOI: https://dx.doi.org/10.1186/s12906-020-2819-7]

39. Jia, Y.; Wu, C.; Kim, Y.S.; Yang, S.O.; Kim, Y.; Kim, J.S.; Lee, S.J.; Yoon, Y.E.; Thach, T.T.; Lee, S.J. et al. A dietary anthocyanin cyanidin-3-O-glucoside binds to PPARs to regulate glucose metabolism and insulin sensitivity in mice. Commun. Biol.; 2020; 3, 514. [DOI: https://dx.doi.org/10.1038/s42003-020-01231-6]

40. Ma, B.; Wu, Y.; Chen, B.; Yao, Y.; Wang, Y.; Bai, H.; Li, C.; Yang, Y.; Chen, Y. Cyanidin-3-O-β-glucoside attenuates allergic airway inflammation by modulating the IL-4Rα-STAT6 signaling pathway in a murine asthma model. Int. Immunopharmacol.; 2019; 69, pp. 1-10. [DOI: https://dx.doi.org/10.1016/j.intimp.2019.01.008] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30660871]

41. Li, J.; Zou, C.; Liu, Y. Amelioration of ovalbumin-induced food allergy in mice by targeted rectal and colonic delivery of cyanidin-3-o-glucoside. Foods; 2022; 11, 1542. [DOI: https://dx.doi.org/10.3390/foods11111542]

42. Hiemori-Kondo, M.; Morikawa, E.; Fujikura, M.; Nagayasu, A.; Maekawa, Y. Inhibitory effects of cya-nidin-3-O-glucoside in black soybean hull extract on RBL-2H3 cells degranulation and passive cutaneous anaphylaxis reaction in mice. Int. Immunopharmacol.; 2021; 94, 107394. [DOI: https://dx.doi.org/10.1016/j.intimp.2021.107394] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33582590]

43. Sánchez-Rangel, J.C.; Benavides, J.; Heredia, J.B.; Cisneros-Zevallos, L.; Jacobo-Velázquez, D.A. The Folin–Ciocalteu assay revisited: Improvement of its specificity for total phenolic content determination. Anal. Methods; 2013; 5, pp. 5990-5999. [DOI: https://dx.doi.org/10.1039/c3ay41125g]

44. Benítez, V.; Mollá, E.; Martín-Cabrejas, M.A.; López-Andréu, F.J.; Downes, K.; Terry, L.A.; Esteban, R.M. Study of bioactive compound content in different onion sections. Plant Foods Hum. Nutr.; 2011; 66, pp. 48-57. [DOI: https://dx.doi.org/10.1007/s11130-011-0212-x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21318305]

45. Kim, J.H.; Park, J.H.; Park, S.D.; Choi, S.Y.; Seong, J.H.; Moon, K.D. Preparation and antioxidant activity of health drink with extract powders from safflower (Carthamus tinctorius L.) seed. Korean J. Food Sci. Technol.; 2002; 34, pp. 617-624.

46. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free. Radic. Biol. Med.; 1999; 26, pp. 1231-1237. [DOI: https://dx.doi.org/10.1016/S0891-5849(98)00315-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10381194]

47. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem.; 1996; 239, pp. 70-76. [DOI: https://dx.doi.org/10.1006/abio.1996.0292] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8660627]

48. Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem.; 2001; 49, pp. 4619-4626. [DOI: https://dx.doi.org/10.1021/jf010586o]

49. Ji, Y.M.; Kim, M.Y.; Lee, S.H.; Jang, G.Y.; Li, M.; Yoon, N.; Kim, K.M.; Lee, J.; Jeong, H.S. Effects of acidic treatments for anthocyanin and proanthocyanidin extraction on black bean (Glycine max Merrill.). J. Korean Soc. Food Sci. Nutr.; 2015; 44, pp. 1594-1598. [DOI: https://dx.doi.org/10.3746/jkfn.2015.44.10.1594]

50. Guo, L.; Qiao, J.; Gong, C.; Wei, J.; Li, J.; Zhang, L.; Qin, D.; Huo, J. C3G quantified method verification and quantified in blue honeysuckle (Lonicera caerulea L.) using HPLC–DAD. Heliyon; 2023; 9, e14685. [DOI: https://dx.doi.org/10.1016/j.heliyon.2023.e14685] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37035373]

51. Gołba, M.; Sokół-Łętowska, A.; Kucharska, A.Z. Health properties and composition of honeysuckle berry Lonicera caerulea L. An update on recent studies. Molecules; 2020; 25, 749. [DOI: https://dx.doi.org/10.3390/molecules25030749] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32050498]

52. Nagata, K.; Araumi, S.; Ando, D.; Ito, N.; Ando, M.; Ikeda, Y.; Nishiyama, C. Kaempferol suppresses the activation of mast cells by modulating the expression of FcεRI and SHIP1. Int. J. Mol. Sci.; 2023; 24, 5997. [DOI: https://dx.doi.org/10.3390/ijms24065997]

53. Lee, J.Y.; Park, S.H.; Jhee, K.H.; Yang, S.A. Tricin isolated from enzyme-treated Zizania 54. latifolia extract inhibits IgE-mediated allergic reactions in RBL-2H3 cells by targeting the Lyn/Syk pathway. Molecules; 2020; 25, 2084. [DOI: https://dx.doi.org/10.3390/molecules25092084]

54. Min, S.Y.; Park, C.H.; Yu, H.W.; Park, Y.J. Anti-inflammatory and anti-allergic effects of saponarin and its impact on signaling pathways of RAW 264.7, RBL-2H3, and HaCaT Cells. Int. J. Mol. Sci.; 2021; 22, 8431. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34445132]

55. Comhair, S.A.; Erzurum, S.C. Redox control of asthma: Molecular mechanisms and therapeutic opportunities. Antioxid. Redox Signal.; 2010; 12, pp. 93-124. [DOI: https://dx.doi.org/10.1089/ars.2008.2425] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19634987]

56. Qu, J.; Li, Y.; Zhong, W.; Gao, P.; Hu, C. Recent developments in the role of reactive oxygen species in allergic asthma. J. Thorac. Dis.; 2017; 9, E32. [DOI: https://dx.doi.org/10.21037/jtd.2017.01.05] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28203435]

57. Munteanu, I.G.; Apetrei, C. Analytical methods used in determining antioxidant activity: A review. Int. J. Mol. Sci.; 2021; 22, 3380. [DOI: https://dx.doi.org/10.3390/ijms22073380] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33806141]

58. Raudonė, L.; Liaudanskas, M.; Vilkickytė, G.; Kviklys, D.; Žvikas, V.; Viškelis, J.; Viškelis, P. Phenolic profiles, antioxidant activity and phenotypic characterization of Lonicera caerulea L. berries, cultivated in Lithuania. Antioxidants; 2021; 10, 115. [DOI: https://dx.doi.org/10.3390/antiox10010115] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33467507]

59. Holgate, S.T.; Polosa, R. Treatment strategies for allergy and asthma. Nat. Rev. Immunol.; 2008; 8, pp. 218-230. [DOI: https://dx.doi.org/10.1038/nri2262] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18274559]

60. Torres-Atencio, I.; Ainsua-Enrich, E.; De Mora, F.; Picado, C.; Martín, M. Prostaglandin E2 prevents hyperosmolar-induced human mast cell activation through prostanoid receptors EP2 and EP4. PLoS ONE; 2014; 9, e110870.

61. Je, I.G.; Choi, H.G.; Kim, H.H.; Lee, S.; Choi, J.K.; Kim, S.W.; Kim, S.H. Inhibitory effect of 1, 2, 4, 5-tetramethoxybenzene on mast cell-mediated allergic inflammation through suppression of IκB kinase complex. Toxicol. Appl. Pharmacol.; 2015; 287, pp. 119-127. [DOI: https://dx.doi.org/10.1016/j.taap.2015.05.006] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25981167]

62. Ott, V.L.; Cambier, J.C. Activating and inhibitory signaling in mast cells: New opportunities for therapeutic intervention?. J. Allergy Clin. Immunol.; 2000; 106, pp. 429-440. [DOI: https://dx.doi.org/10.1067/mai.2000.109428] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10984360]

63. Gilfillan, A.M.; Rivera, J. The tyrosine kinase network regulating mast cell activation. Immunol. Rev.; 2009; 228, pp. 149-169. [DOI: https://dx.doi.org/10.1111/j.1600-065X.2008.00742.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19290926]

64. Guo, X.; Gerl, R.E.; Schrader, J.W. Defining the involvement of p38α MAPK in the production of anti-and proinflammatory cytokines using an SB 203580-resistant form of the kinase. J. Biol. Chem.; 2003; 278, pp. 22237-22242. [DOI: https://dx.doi.org/10.1074/jbc.M300847200]

65. Alam, R.; Gorska, M.M. Mitogen-activated protein kinase signalling and ERK1/2 bistability in asthma. Clin. Exp. Allergy; 2011; 41, pp. 149-159. [DOI: https://dx.doi.org/10.1111/j.1365-2222.2010.03658.x]

66. Gutowska-Ślesik, J.; Samoliński, B.; Krzych-Fałta, E. The increase in allergic conditions based on a review of literature. Adv. Dermatol. Allergol./Postępy Dermatol. I Alergol.; 2023; 40, pp. 1-7. [DOI: https://dx.doi.org/10.5114/ada.2022.119009]

67. Stróżek, J.; Samoliński, B.; Kłak, A.; Gawińska-Drużba, E.; Izdebski, R.; Krzych-Fałta, E.; Raciborski, F. The indirect costs of allergic diseases. Int. J. Occup. Med. Environ. Health; 2019; 32, pp. 281-290. [DOI: https://dx.doi.org/10.13075/ijomeh.1896.01275] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31180070]

68. OCHMIAN, I.D.; Skupien, K.; Grajkowski, J.; Smolik, M.; Ostrowska, K. Chemical composition and physical characteristics of fruits of two cultivars of blue honeysuckle (Lonicera caerulea L.) in relation to their degree of maturity and harvest date. Not. Bot. Horti Agrobot. Cluj Napoca; 2012; 40, pp. 155-162. [DOI: https://dx.doi.org/10.15835/nbha4017314]

69. Rop, O.; Řezníček, V.; Mlček, J.; Juríková, T.; Balík, J.; Sochor, J.; Kramářová, D. Antioxidant and radical oxygen species scavenging activities of 12 cultivars of blue honeysuckle fruit. Hortic. Science; 2011; 38, pp. 63-70. [DOI: https://dx.doi.org/10.17221/99/2010-HORTSCI]

70. Rupasinghe, H.V.; Yu, L.J.; Bhullar, K.S.; Bors, B. Haskap (Lonicera caerulea): A new berry crop with high antioxidant capacity. Can. J. Plant Sci.; 2012; 92, pp. 1311-1317. [DOI: https://dx.doi.org/10.4141/cjps2012-073]

71. Gawroński, J.; Żebrowska, J.; Pabich, M.; Jackowska, I.; Kowalczyk, K.; Dyduch-Siemińska, M. Phytochemical characterization of blue honeysuckle in relation to the genotypic diversity of Lonicera sp. Appl. Sci.; 2020; 10, 6545. [DOI: https://dx.doi.org/10.3390/app10186545]

72. Bakowska-Barczak, A.M.; Marianchuk, M.; Kolodziejczyk, P. urvey of bioactive components in Western Canadian berries. Can. J. Physiol. Pharmacol.; 2007; 85, pp. 1139-1152. [DOI: https://dx.doi.org/10.1139/Y07-102]

73. Kedare, S.B.; Singh, R.P. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol.; 2011; 48, pp. 412-422. [DOI: https://dx.doi.org/10.1007/s13197-011-0251-1] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23572765]

74. Alam, M.N.; Bristi, N.J.; Rafiquzzaman, M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J.; 2013; 21, pp. 143-152. [DOI: https://dx.doi.org/10.1016/j.jsps.2012.05.002]

75. Antolovich, M.; Prenzler, P.D.; Patsalides, E.; McDonald, S.; Robards, K. Methods for testing antioxidant activity. Analyst; 2002; 127, pp. 183-198. [DOI: https://dx.doi.org/10.1039/b009171p] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11827390]

76. Cao, G.; Alessio, H.M.; Cutler, R.G. Oxygen-radical absorbance capacity assay for antioxidants. Free. Radic. Biol. Med.; 1993; 14, pp. 303-311. [DOI: https://dx.doi.org/10.1016/0891-5849(93)90027-R] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8458588]

77. Senica, M.; Stampar, F.; Mikulic-Petkovsek, M. Blue honeysuckle (Lonicera cearulea L. subs. edulis) berry; A rich source of some nutrients and their differences among four different cultivars. Sci. Hortic.; 2018; 238, pp. 215-221.

78. Khattab, R.; Brooks, M.S.L.; Ghanem, A. Phenolic analyses of haskap berries (Lonicera caerulea L.): Spectrophotometry versus high performance liquid chromatography. Int. J. Food Prop.; 2016; 19, pp. 1708-1725. [DOI: https://dx.doi.org/10.1080/10942912.2015.1084316]

79. Gorzelany, J.; Basara, O.; Kapusta, I.; Paweł, K.; Belcar, J. Evaluation of the Chemical Composition of Selected Varieties of L. caerulea var. kamtschatica and L. caerulea var. emphyllocalyx. Molecules; 2023; 28, 2525. [DOI: https://dx.doi.org/10.3390/molecules28062525]

80. Myjavcová, R.; Marhol, P.; Křen, V.; Šimánek, V.; Ulrichová, J.; Palíková, I.; Papoušková, B.; Lemr, K.; Bednář, P. Analysis of anthocyanin pigments in Lonicera (Caerulea) extracts using chromatographic fractionation followed by microcolumn liquid chromatography-mass spectrometry. J. Chromatogr. A; 2010; 1217, pp. 7932-7941. [DOI: https://dx.doi.org/10.1016/j.chroma.2010.05.058] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21111888]

81. Fang, X.; Lang, Y.; Wang, Y.; Mo, W.; Wei, H.; Xie, J.; Yu, M. Shp2 activates Fyn and Ras to regulate RBL-2H3 mast cell activation following FcεRI aggregation. PLoS ONE; 2012; 7, e40566. [DOI: https://dx.doi.org/10.1371/journal.pone.0040566] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22802969]

82. Kanagy, W.K.; Cleyrat, C.; Fazel, M.; Lucero, S.R.; Bruchez, M.P.; Lidke, K.A.; Wilson, B.S.; Lidke, D.S. Docking of Syk to FcεRI is enhanced by Lyn but limited in duration by SHIP1. Mol. Biol. Cell; 2022; 33, ar89. [DOI: https://dx.doi.org/10.1091/mbc.E21-12-0603] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35793126]

83. Park, Y.H.; Kim, D.K.; Kim, H.W.; Kim, H.S.; Lee, D.; Lee, M.B.; Choi, W.S. Repositioning of anti-cancer drug candidate, AZD7762, to an anti-allergic drug suppressing IgE-mediated mast cells and allergic responses via the inhibition of Lyn and Fyn. Biochem. Pharmacol.; 2018; 154, pp. 270-277. [DOI: https://dx.doi.org/10.1016/j.bcp.2018.05.012] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29777684]

84. Quarto, R.; Kinet, J.P.; Metzger, H. Coordinate synthesis and degradation of the α-, β-and γ-subunits of the receptor for immunoglobulin E. Mol. Immunol.; 1985; 22, pp. 1045-1051. [DOI: https://dx.doi.org/10.1016/0161-5890(85)90107-5]

85. Yamaguchi, M.; Lantz, C.S.; Oettgen, H.C.; Katona, I.M.; Fleming, T.; Miyajima, I.; Kinet, J.-P.; Galli, S.J. IgE enhances mouse mast cell FcεRI expression in vitro and in vivo: Evidence for a novel amplification mechanism in IgE-dependent reactions. J. Exp. Med.; 1997; 185, pp. 663-672. [DOI: https://dx.doi.org/10.1084/jem.185.4.663] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/9034145]

86. Mócsai, A.; Ruland, J.; Tybulewicz, V.L. The SYK tyrosine kinase: A crucial player in diverse biological functions. Nat. Rev. Immunol.; 2010; 10, pp. 387-402. [DOI: https://dx.doi.org/10.1038/nri2765] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20467426]

87. Sada, K.; Takano, T.; Yanagi, S.; Yamamura, H. Structure and function of Syk protein-tyrosine kinase. J. Biochem.; 2001; 130, pp. 177-186. [DOI: https://dx.doi.org/10.1093/oxfordjournals.jbchem.a002970] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11481033]

88. Nishizuka, Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature; 1984; 308, pp. 693-698. [DOI: https://dx.doi.org/10.1038/308693a0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/6232463]

89. Yang, Y.; Kim, S.C.; Yu, T.; Yi, Y.S.; Rhee, M.H.; Sung, G.H.; Yoo, B.C.; Cho, J.Y. Functional roles of p38 mitogen-activated protein kinase in macrophage-mediated inflammatory responses. Mediat. Inflamm.; 2014; 352371, 17. [DOI: https://dx.doi.org/10.1155/2014/352371] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24771982]

90. Cohen, R.; Holowka, D.A.; Baird, B.A. Real-time imaging of Ca²⁺ mobilization and degranulation in mast cells. Mast Cells Methods Protoc.; 2015; 1220, pp. 347-363.

91. Theoharides, T.C.; Alysandratos, K.D.; Angelidou, A.; Delivanis, D.A.; Sismanopoulos, N.; Zhang, B.; Asadi, S.; Vasiadi, M.; Weng, Z.; Kalogeromitros, D. et al. Mast cells and inflammation. Biochim. Biophys. Acta Mol. Basis Dis.; 2012; 1822, pp. 21-33. [DOI: https://dx.doi.org/10.1016/j.bbadis.2010.12.014] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21185371]

92. Wagner, A.; Alam, S.B.; Kulka, M. The effects of age, origin, and biological sex on rodent mast cell (BMMC and MC/9) and basophil (RBL-2H3) phenotype and function. Cell Immunol.; 2023; 391, 104751. [DOI: https://dx.doi.org/10.1016/j.cellimm.2023.104751] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37480823]

93. Kong, Z.L.; Sudirman, S.; Lin, H.J.; Chen, W.N. In vitro anti-inflammatory effects of curcumin on mast cell-mediated allergic responses via inhibiting FcεRI protein expression and protein kinase C delta translocation. Cytotechnology; 2020; 72, pp. 81-95. [DOI: https://dx.doi.org/10.1007/s10616-019-00359-6]

94. Ma, J.; Tong, P.; Chen, Y.; Wang, Y.; Ren, H.; Gao, Z.; Yue, T.; Long, F. The inhibition of pectin oligosaccharides on degranulation of RBL-2H3 cells from apple pectin with high hydrostatic pressure assisted enzyme treatment. Food Chem.; 2022; 371, 131097. [DOI: https://dx.doi.org/10.1016/j.foodchem.2021.131097] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34537607]

95. Sanak, M.; Potaczek, D.P.; Nizankowska-Mogilnicka, E.; Szczeklik, A. Genetic Variability of the High-affinity IgE Receptor α Subunit (Fc ε RI α) is Related to Total Serum IgE levels in Allergic Subjects. Allergol. Int.; 2007; 56, pp. 397-401. [DOI: https://dx.doi.org/10.2332/allergolint.R-07-145]

96. Acevedo, N.; Alashkar Alhamwe, B.; Caraballo, L.; Ding, M.; Ferrante, A.; Garn, H.; Garssen, J.; Hii, C.S.; Irvine, J.; Llinás-Caballero, K. et al. Perinatal and early-life nutrition, epigenetics, and allergy. Nutrients; 2021; 13, 724. [DOI: https://dx.doi.org/10.3390/nu13030724] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33668787]