Compound 48/80, anti-dinitrophenyl (DNP) IgE, DNP-human serum albumin (HSA), phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 and disodium cromoglycate (DSCG) were purchased from Sigma (St. Louis, MO, USA). The human mast cell line (HMC-1) was grown in Iscove’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum at 37°C in 5% CO₂. HMC-1 cells of passage 4–8 were used in all the experiments.

The original stock of male imprinting control region (ICR) mice (6 weeks of age) was purchased from Dae Han Bio Link Co., Ltd (Eumsung-Gun, Korea). The animals were housed 5 per cage in a laminar air flow room maintained under a temperature of 22±2°C and a relative humidity of 55±5% throughout the study. The care and treatment of the mice were in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at Kyungpook National University.

D. kaki was supplied from the Korea National Arboretum. A voucher specimen (no. WSP-10-38) was deposited at the herbarium of the College of Pharmacy, Woosuk University, Jeonju, Korea. D. kaki (600 g) was shade dried and powdered. It was then extracted 3 times with purified water (500 ml) at 70°C for 5 h in a water bath and filtered through a Whatman no. 1 filter. The extracts were combined and evaporated. The yield of dried extract from the crude materials was approximately 4.16% (w/w). The dried extract of AEDK was dissolved in saline or Tyrode buffer and was filtered using a 0.45 μm syringe filter. To isolate the active components responsible for the anti-allergic and anti-inflammatory effects of AEDK, the resultant water extract (70 g) was successively partitioned as chloroform, ethyl acetate, n-butanol and water soluble fractions. The ethyl acetate soluble fraction exhibited the highest anti-allergic activity. The ethyl acetate soluble fraction was chromatographed on a Sephadex LH-20 column (MeOH) (GE Healthcare Bio-Sciences, Uppsala, Sweden) to yield 5 fractions (E1–E5). E2 was subjected to chromatography on a Sephadex LH-20 gel column (MeOH) to yield compound 1 (25 mg). The molecular formulae of compound 1 were characterized by nuclear magnetic resonance (NMR). By comparing the NMR data and literature values (18,19), compound 1 was identified as a catechin.

The degranulation of mast cells was monitored by measuring the release of histamine and β-hexosaminidase. The histamine content in serum and HMC-1 cells was measured using the o-phthaldialdehyde spectrofluorometric procedure as previously described (20). HMC-1 cells (1×10⁶ cells/ml) were pre-incubated with AEDK for 30 min, and then incubated for 30 min with PMA (20 nM) and calcium ionophore A23187 (PMACI) (1 μM) as previously described (21). The release of β-hexosaminidase was measured as previously described (22). In brief, the HMC-1 cells were treated with AEDK at 37°C for 30 min. The cells were sensitized with PMACI for 30 min and placed on ice for 10 min to terminate the reaction.

The cAMP level was measured as previously described (4). The intracellular calcium concentration was measured with the use of the fluorescence indicator Fluo-3/AM (Molecular Probes, Eugene, OR, USA). The HMC-1 cells were pre-incubated with Fluo-3/AM for 30 min at 37°C. After washing the dye from the cell surface, the cells were treated with AEDK for 5 min prior to the addition of PMACI. They were excited at 488 nm, the emission was filtered with 515 nm by a flow cytometer (BD Biosciences Pharmingen, San Diego, CA, USA) and visualized under a fluorescence microscope (Olympus BX51; Olympus, Center Valley, PA, USA).

Total cellular RNA was isolated from the cells (1×10⁶ cells/well in a 24-well plate) following stimulation with PMACI with or without AEDK for 4 h. Real-time polymerase chain reaction (PCR) was used to analyze the mRNA expression of TNF-α, IL-1β and β-actin (internal control) as previously described (23). The band intensity was normalized to that of β-actin in the same sample. The secretion of TNF-α and IL-1β was measured by ELISA as previously described (4). The HMC-1 cells were cultured in medium and resuspended in Tyrode buffer A. The cells were sensitized with PMACI for 8 h in the absence or presence of AEDK.

The samples were electrophoresed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as previously described (24) and then transferred onto a nitrocellulose membrane. Nuclear and cytosolic p65 NF-κB and IκBα were assayed using anti-NF-κB (p65) and anti-IκBα antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Luciferase activity was measured as previously described (21). The HMC-1 cells were seeded at 2×10⁶ in a 6-well plate 1 day prior to transient transfection. After the transfected cells were incubated for 18 h, they were stimulated with PMACI at 37°C for 2 h. The cells were pre-treated with AEDK for 30 min prior to stimulation with PMACI. Luciferase activity was determined using a luminometer.

Mice were administered an intraperitoneal injection [8 mg/kg of body weight (BW)] of the mast cell degranulator, compound 48/80. AEDK was dissolved in saline and administered intraperitoneally (1–100 mg/kg BW) 1 h prior to the injection of compound 48/80 (n=10/group). Mortality was monitored for 1 h after the induction of anaphylactic shock. After the mortality test, blood was obtained from the heart of each mouse to measure the serum histamine content.

An IgE-dependent cutaneous reaction was carried out as previously described (23). The mice were injected intradermally with 0.5 μg of anti-DNP IgE. After 48 h, each mouse (n=10/group) received an injection of 1 μg of DNP-HSA containing 4% Evans blue (1:4) via the tail vein. Thirty minutes after the challenge, the mice were sacrificed and the dorsal skin (diameter, 1 cm) was removed in order to measure the pigmented area. The amount of dye was then determined colorimetrically.

Statistical analyses were performed using SAS statistical software (SAS Institute, Cary, NC). The effects of treatment were analyzed using an analysis of variance, followed by Duncan’s multiple range tests. A P-value <0.05 was considered to indicate a statistically significant difference.

We determined the effects of AEDK on the degranulation of mast cells. The release of histamine and β-hexosaminidase from activated mast cells is a hallmark of degranulation. HMC-1 cells released a high level of histamine when stimulated with PMACI (Fig. 1A). When pre-treated with AEDK (1–100 μg/ml) for 30 min, histamine levels were dose-dependently inhibited in the PMACI-stimulated HMC-1 cells. To confirm the inhibitory effects of AEDK on the degranulation of mast cells, we investigated the effects of AEDK on the release of β-hexodaminidase (Fig. 1B). The results revealed that 10 to 100 μg/ml AEDK significantly decreased the PMACI-stimulated release of β-hexodaminidase from HMC-1 cells. The concentration and duration of AEDK treatment used in these experiments had no significant effect on the cell viability of HMC-1 cells (data not shown).

To investigate the mechanisms responsible for the reduction in the levels of histamine following treatment with AEDK, we assayed the cAMP and intracellular calcium levels. When the HMC-1 cells were incubated with AEDK (100 μg/ml), the cAMP content increased within 15 sec and remained at relatively high levels for 120 sec (Fig. 2A). Calcium movements across the membranes of mast cells are critical to histamine release (25). To further investigate the mechanisms responsible for the reduction of the release of histamine by AEDK, we assayed the intracellular calcium levels. When the HMC-1 cells were stimulated with PMACI, the intracellular calcium level was significantly elevated (Fig. 2B). However, the pre-incubation of HMC-1 cells with AEDK (100 μg/ml) decreased the intracellular calcium level induced by PMACI. The level of intracellular calcium was also depicted by the relative fluorescence intensity (Fig. 2C).

We investigated the inhibitory effects of AEDK on the expression of pro-inflammatory cytokines, such as TNF-α and IL-1β. Previously, we reported that the gene expression of TNF-α and IL-1β peaked 4 h after treatment with PMACI (26). Consequently, the HMC-1 cells were stimulated with PMACI for 4 h following pre-treatment with AEDK for 30 min. Fig. 3A illustrates that the expression of pro-inflammatory cytokines was inhibited by AEDK. To confirm the correlation of mRNA expression with protein production, we evaluated the secretion of TNF-α and IL-1β with ELISA. When the HMC-1 cells were stimulated with PMACI for 8 h, the secretion of cytokines was markedly induced. The secretion of TNF-α and IL-1β was significantly inhibited by AEDK (10 and 100 μg/ml) in the PMACI-stimulated HMC-1 cells (Fig. 3B).

To investigate the intracellular mechanisms responsible for the inhibitory effects of AEDK on the expression of pro-inflammatory cytokines, we examined the effects of AEDK on the activation of the transcription factor, NF-κB. NF-κB is an important transcriptional regulator of inflammatory cytokines and plays a crucial role in immune and inflammatory responses (4). The stimulation of HMC-1 cells with PMACI induced the nuclear translocation of p65 NF-κB and the degradation of IκBα. AEDK inhibited the PMACI-induced nuclear translocation of NF-κB and the degradation of IκBα (Fig. 4A). To confirm the inhibitory effects of AEDK on NF-κB activation, we used an NF-κB-dependent gene reporter assay. The HMC-1 cells were transiently transfected with a NF-κB-luciferase reporter construct or an empty vector. Exposure of the cells to PMACI increased the luciferase activity in the cells transfected with the NF-κB-luciferase reporter construct (Fig. 4B). AEDK (100 μg/ml) significantly reduced the PMACI-induced luciferase activity.

To determine the effects of AEDK on allergic reaction, an in vivo model of systemic anaphylaxis was used. Compound 48/80 (8 mg/kg, BW) was used as a model of induction for a systemic fatal allergic reaction. Groups of mice (n=10/group) were intraperitoneally injected with 200 μl of saline or drugs at various doses, 1 h before the intraperitoneal injection of compound 48/80. After the intraperitoneal injection of compound 48/80, the mice were monitored for 1 h, after which the mortality rate was determined as the number of dead mice ×100/total number of experimental mice. All mice underwent fatal shock after the injection of compound 48/80. We compared the anti-anaphylactic effects of AEDK with DSCG, a known anti-allergic drug. When AEDK and DSCG were intraperitoneally administered, at doses ranging from 1 to 100 mg/kg (BW) for 1 h, the mortality rate was dose-dependently reduced (Table I). The effect of AEDK on the compound 48/80-induced release of histamine in serum was also investigated. AEDK and DSCG were administered 1 h prior to the injection of compound 48/80. The injection of compound 48/80 induced a marked increase in the release of histamine in serum, which was inhibited by treatment with AEDK in a dose-dependent manner (Fig. 5A).

Another way to examine the anaphylactic reaction is to induce PCA. A local extravasation was induced by the local injection of IgE followed by an antigenic challenge. AEDK and DSCG were intraperitoneally administered at 1 to 100 mg/kg (BW) 1 h prior to the antigen challenge. AEDK dose-dependently reduced the IgE-mediated PCA reaction (Fig. 5B). The inhibitory effects of AEDK on systemic and local anaphylaxis were similar to those of DSCG.