The Journal of Allergy and Clinical Immunology
Volume 106, Issue 1 , Pages 159-166, July 2000

Persimmon leaf extract and astragalin inhibit development of dermatitis and IgE elevation in NC/Nga mice☆☆☆

  • Mayumi Kotani, BSc

      Affiliations

    • Research and Development Center, Sunstar Incorporation, Osaka Osaka, Japan
    • ,
  • Motonobu Matsumoto, BSc

      Affiliations

    • Research and Development Center, Sunstar Incorporation, Osaka Osaka, Japan
    • ,
  • Akihito Fujita, BSc

      Affiliations

    • Research and Development Center, Sunstar Incorporation, Osaka Osaka, Japan
    • ,
  • Shinji Higa, MD

      Affiliations

    • Department III of Internal Medicine, Osaka University Medical School, Osaka. Osaka, Japan
    • ,
  • Way Wang, MD, PhD

      Affiliations

    • Department III of Internal Medicine, Osaka University Medical School, Osaka. Osaka, Japan
    • ,
  • Masaki Suemura, MD, PhD

      Affiliations

    • Department III of Internal Medicine, Osaka University Medical School, Osaka. Osaka, Japan
    • ,
  • Tadamitsu Kishimoto, MD, PhD

      Affiliations

    • Department III of Internal Medicine, Osaka University Medical School, Osaka. Osaka, Japan
    • ,
  • Toshio Tanaka, MD, PhD

      Affiliations

    • Department III of Internal Medicine, Osaka University Medical School, Osaka. Osaka, Japan

Received 11 February 2000; received in revised form 16 March 2000; accepted 16 March 2000.

Article Outline

Abstract 

Background: We previously found that persimmon leaf extract contains antiallergic substances that inhibit histamine release by human basophilic cell line KU812 in response to cross-linkage of FcϵRI. Objectives: The purpose of this study was to identify substances in the persimmon leaf extract that are responsible for the effect and to examine their in vivo effects on the allergic mouse model. Methods: HPLC analysis of persimmon leaf extract was done to measure its content. Inhibitory activity of persimmon leaf extract or its major constituent of flavonoids (astragalin) on the histamine release by KU812 cells was examined. To investigate the effects of these substances in vivo, models of passive cutaneous anaphylaxis and atopic dermatitis mice (NC/Nga) were used. Results: Persimmon leaf extract or astragalin inhibited histamine release from KU812 in response to cross-linkage of FcϵRI. Oral intake of both substances dose dependently inhibited passive cutaneous reactions. Moreover, oral administration of these substances to NC/Nga atopic dermatitis–model mice led to a striking suppression of the development of dermatitis, scratching behavior, and serum IgE elevation. Histologic analyses revealed that infiltration of inflammatory cells, especially degranulated mast cells, thickening of the epidermis, and prominent hyperkeratosis, were significantly reduced. Immunologic studies showed that the capacity of spleen T cells to produce both IL-4 and IL-13, but not IFN-γ, was downregulated by means of oral intake of these substances. Conclusion: This study demonstrates a novel activity of astragalin and the dramatic effect of persimmon leaf extract and astragalin on atopic dermatitis–model mice. (J Allergy Clin Immunol 2000;106:159-66.)

Keywords:  Persimmon leaf extract, astragalin, histamine, passive cutaneous reaction, NC/Nga mouse, atopic dermatitis, skin, hypersensitivity, IgE

Abbreviations:  FcϵRI , High-affinity IgE receptor, PCA , Passive cutaneous anaphylaxis

 

Atopic dermatitis is a common disease and has been recently increasing in industrialized countries.1 Recent immunologic analyses of the pathogenesis of this disease have revealed that activated mast cells and excess of differentiated TH2 cells through chemical mediators and cytokines might play major roles in the development of dermatitis and IgE synthesis.2, 3 Although it has been accepted that topical steroid therapy is crucial for the management of the disease,4, 5 many patients are still dissatisfied with its effectiveness and are worried about chronic use of this drug.

The persimmon Diospyros kaki thunberg (Ebenaceae) grows in China, Korea, and Japan. It was found that tea brewed from the leaves of this persimmon tree had a beneficial effect on hemostasis, diuresis, constipation, and hypotension. Flavonol kaemp-ferol, one of the flavonoids, was found in a hot water extract from persimmon leaves.6 Kaempferol has been reported to inhibit antigen- or ionophore A23187– induced histamine secretion from rat mast cells,7, 8 DNP-BSA antigen–induced hexosaminidase secretion from RBL-2H3 cells,9 and phorbol myristate acetate–induced O2 production from human neutrophils.10 Kaempferol-3-glucoside (astragalin) from persimmon leaves was found to possess hypotensive action through its suppression of angiotensin-converting enzyme activity.11, 12 Kaempferol and astragalin from the white petals of Impatiens balsamina L were shown to have an antipruritic effect on dextran T40–evoked scratching behavior in mice.13 However, so far there is little in the literature on the antiallergic effect of astragalin. In a previous study we showed that persimmon leaf extract inhibited histamine release from human basophilic cell line KU812 and the 24-hour homologous passive cutaneous anaphylaxis (PCA) reaction in mice.14 In the present study we examined the inhibitory effects of persimmon leaf extract and of astragalin, which is considered to be the primary ingredient for the action of persimmon leaf, on a human basophilic cell line KU812 and on NC/Nga atopic dermatitis model mice.

Back to Article Outline

METHODS 

Culture medium 

RPMI-1640 supplemented with 10% FCS (Hyclone Laboratories, Logan, Utah), L -glutamine (2 mmol/L), 2-mercaptoethanol (0.05 mmol/L), penicillin (100 U/mL), and streptomycin (100 μg/mL) was used as the culture medium.

Chemicals 

Persimmon leaf from Shi-chuan, China, was purchased at a retail shop in Japan. Kaempferol-3-glucoside was purchased from Extrasynthese (Genay, France), RPMI-1640 was purchased from Gibco BRL (New York, NY), epinephrine was purchased from Daiichi Pharmaceutical Co Ltd (Tokyo, Japan), histamine EIA kit was purchased from Immunotech A Beckman Coulter Co (Marseille, France), and CRA-1 (anti-FcϵRIα mAb)15 was purchased from Kyokuto Pharmaceutical Industry Co Ltd (Tokyo, Japan). The MF diet and α-corn starch were obtained from Oriental Bioservice Co Ltd (Kyoto, Japan), and A23187 was purchased from Sigma Japan (Tokyo).

Preparation of persimmon leaf extract 

The persimmon leaf was steeped in boiling distilled water for 5 minutes and filtered through a nylon mesh (pore size, 40 μm). After centrifugation at 5000 rpm for 20 minutes at 4°C, the supernatant of the persimmon leaf extract was freeze-dried. The contaminated endotoxin level in persimmon leaf extract and astragalin was less than 1 ng/mL.

HPLC quantitative analysis of astragalin in persimmon leaf extract 

Astragalin was quantified by means of RP-HPLC in a separative column of YMC-pack ODS-A A-312 (6 × 150 mm; particle size, 5 μm) with water/acetonitrile/2-propanol (180:38:2) as the mobile phase, a flow rate of 1.0 mL/min at room temperature, and UV detection at 350 nm. Astragalin quantification was carried out in duplicate and determined by comparison with a standard astragalin absorption curve.

Histamine release assay 

KU812 cells (human basophilic leukemia cells)16 were grown in RPMI-1640 supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 in air at 37°C. KU812 cells were suspended in a histamine release buffer (30 mmol/L TRIS-HCl [pH 7.6], 120 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 0.03% BSA). The first incubation of 100 μL of KU812 cells (105 cells/mL) was performed with 100 μL of persimmon leaf extract (0.02%, 0.01%) and astragalin (0.0001%, 0.00005%) in an ice bath for 30 minutes. After centrifugation at 3000 rpm for 5 minutes at 4°C, 200 μL of histamine release buffer was added to the cells removed from the supernatant, which was then incubated with 15 μg/mL CRA-1 at 37°C for 30 minutes. After centrifugation at 3000 rpm for 5 minutes at 4°C, the histamine content of the supernatant and cell lysate was measured by means of ELISA assay. Positive and negative controls were used to indicate histamine content obtained with or without CRA-1.

PCA reaction 

PCA reaction was evaluated according to the previous method.17 Briefly, 6-week-old male ddY mice were obtained from Japan SLC (Shizuoka, Japan) and housed at 23°C ± 3°C in 55% ± 15% humidity with 12 hour-12 hour light-dark cycle (light on 7 AM -7 PM ). After preliminary breeding for 3 days, the mice were divided into 6 groups of 3 each. Twenty microliters of anti-DNP IgE antibody (10 μg/mL; Yamasa Shouyu Co Ltd, Chiba, Japan) was intracutaneously injected into the ears of ddY mice. Twenty-four hours after the sensitization, persimmon leaf extract (100 or 250 mg/kg) and astragalin (0.6 or 1.5 mg/kg) were orally administered by using a metal stomach tube. To the control group and the no treatment group, distilled water was orally administered in the same manner. After 1 hour, mice were challenged with 100 μL of DNP-BSA (1 mg/mL) intravenously. Fifteen minutes thereafter, the thickness of the ear was measured with a thickness gauge 3 times. The ear swelling was calculated according to the following equation: Ear swelling (mm) = Ear thickness after 15 minutes intravenously – Ear thickness after 0 minutes intravenously

Continuous feeding of persimmon leaf extract or astragalin in NC/Nga mice and evaluation of the development of dermatitis 

Four-week-old NC/Nga mice (conventional grade) were obtained from Japan SLC (Shizuoka, Japan) and housed at 23°C ± 3°C in 55% ± 15% humidity with 12 hour-12 hour light-dark cycle (light on 7 AM -7 PM ). After preliminary breeding for 1 week, the mice were divided into 3 groups of 5 each and provided with one of the following diets ad libitum: control diet (MF diet, Oriental Inc), persimmon leaf extract diet (MF diet containing 0.125% persimmon leaf extract, 250 mg/kg), or astragalin diet (control diet plus 0.00075% astragalin, 1.5 mg/kg) during the 8-week experiment. The severity of dermatitis was assessed once a week with the following scoring system. Symptoms were evaluated by skin dryness, eruption, and wound on the 3 parts of the body (ie, ear, face, and head and back). Each symptom was graded from 0 to 3 (no symptoms, 0; mild, 1; moderate, 2; and severe, 3). Blood was taken once every 2 weeks from the eyes, and the serum IgE level was measured by means of an enzyme immunoassay with mAbs (Yamasa Shoyu Co Ltd, Chiba, Japan). The serum levels of immunoglobulins, including IgM, IgG1, IgG2a, IgG2b, and IgG3 at the age of 13 weeks, were also measured by using the Mab-Based Mouse Ig Isotyping kit (PharMingen, San Diego, Calif). Serum was obtained by centrifugation at 3000 rpm for 5 minutes at 4°C and stored at –80°C until use. The frequency of scratching behavior of 13-week-old NC/Nga mice, such as scratching of the nose, ears, and dorsal skin with the hind paws, was measured during a 20-minute period. Licking of the belly and dorsal skin during grooming was disregarded. Each occurrence of scratching of the head, neck, dorsal skin, ears, eyes, and nose was scored to obtain the maximum score.

Histologic study 

Whole ears of the mice at 13 weeks of age were fixed in a formalin solution. A block of the skin was removed and embedded in paraffin, cut in 10-μm sections, and stained with hematoxylin and eosin or toluidine blue.

Preparation of spleen cells 

Cell suspensions were prepared from spleens. Red blood cells were lysed with the ammonium-chloride potassium carbonate buffer, and nucleated cells were washed 3 times and resuspended in the culture medium.

FACS analysis 

Cells were stained with FITC anti-CD3, phycoerythrin anti-CD4, or FITC-anti-B220 antibody (Pharmingen Inc, San Diego, Calif) for 1 hour at 4°C. After washing, the number of positive cells was determined by cytometric analysis on a FACScalibar (Becton Dickinson and Co, Mountain View, Calif) by using PI gating to limit the analysis to living cells.

Lymphokine assays 

Spleen cells (2 × 106/mL) were cultured with soluble anti-CD3 (1 μg/mL) plus anti-CD28 (1 μg/mL) antibodies (Pharmingen) in a 24-well Costar plate (Corning Inc, Cambridge, Mass). Supernatants were collected at 48 hours, and IL-4, IL-13, and IFN-γ concentrations were measured with a 2-site ELISA (Pharmingen).

T-cell proliferation 

Spleen cells (106 cells/mL) were incubated in 96-well, flat-bottom, microtiter plates (Costar, Cambridge, Mass) with or without anti-CD3 (1 μg/mL) plus anti-CD28 (1 μg/mL) antibodies for 3 days in triplicate. For the last 8 hours of culture, 0.5 μCi per well of tritiated (3H) thymidine was added. The cells were harvested onto filters, and 3H-thymidine incorporation was determined by using liquid scintillation spectroscopy.

Statistics 

The Student t test was used for the statistical analysis.

Back to Article Outline

RESULTS 

Freeze-dried extract was obtained from persimmon leaf (0.004 g from 1 mL of persimmon leaf and 0.06 g from 1 g of persimmon leaf). Astragalin (Fig 1) content was determined by means of HPLC at 0.59 g of astragalin per 100 g of persimmon leaf extract (Fig 1).

  • View full-size image.
  • Fig. 1. 

    Chemical structure of astragalin and chromatogram of persimmon leaf extract monitored by UV detector. HPLC conditions: column, YMC-pack ODS-A A-312; mobile phase, water/acetonitrile/2-propanol (180:38:2); flow rate, 1.0 mL/min; detection wavelength, 350 nm.

Both persimmon leaf extract and astragalin inhibit histamine release from human basophilic cell line KU812 in response to Fc ϵ RI cross-linkage 

To examine whether this material could inhibit histamine release from basophilic cells, human cell line KU812 was used. The cross-linkage of FcϵRI by the anti-FcϵRIα chain antibody CRA-1 in KU812 cells induced histamine release as previously demonstrated.14 One representative result of 4 experiments shown in Fig 2 demonstrated that spontaneous secretion of histamine content in the supernatants was 2.75 ± 0.10 nmol/L and that stimulation generated 11.08 ± 1.13 nmol/L of histamine in the supernatants.

  • View full-size image.
  • Fig. 2. 

    Inhibitory effect of persimmon leaf extract and astragalin on histamine release from KU812 cells stimulated with anti-Fcϵ receptor mAb (CRA-1). Cells (104 cells) were at first incubated with persimmon leaf extract (final concentrations, 0.01% and 0.005%) and astragalin (final concentrations, 0.00005% and 0.000025%) on ice for 30 minutes and incubated with 15 μg/mL CRA-1 at 37°C for 30 minutes. Histamine content of the supernatant was measured by means of ELISA. Positive and negative controls indicate histamine content with or without CRA-1. Values represent the mean ± SD of 3 determinations. *P < .05 and **P < .01; significantly different from positive control.

Preincubation of KU812 with epinephrine suppressed its secretion by 40% to 60% in a dose-dependent fashion in every experiment (data not shown). The presence of persimmon leaf extract and astragalin consistently inhibited histamine release, and in this experiment the following inhibition of the release was observed: 38% ± 12% (P < .05) with 0.01% and 33% ± 9% (P < .05) with 0.005% persimmon leaf extract; 40% ± 2% (P < .01) with 0.00005% (1.11 μmol/L) and 25% ± 14% with 0.000025% astragalin.

Effect of oral administration of persimmon leaf extract or astragalin on the passive cutaneous anaphylaxis in mice 

To investigate whether persimmon leaf extract or its major flavonoid astragalin might show antiallergic effects in vivo, a model of PCA was used. ddY mice were injected subcutaneously into ears by 20 μL of anti-DNP IgE antibody (10 μg/mL), and 24 hours later they were orally administered with persimmon leaf extract or astragalin. Thickness of the ear 15 minutes after antigen challenge with DNP-BSA was measured. Fig 3 shows that oral intake of persimmon leaf extract or astragalin inhibits the PCA reaction in a dose-dependent fashion.

  • View full-size image.
  • Fig. 3. 

    Inhibition of PCA reaction by means of persimmon leaf extract or astragalin in ddY mice. ddY mice were injected with anti-DNP IgE antibody. Persimmon leaf extract or astragalin was orally administered into mice, and 1 hour later, DNA-BSA was challenged. Fifteen minutes later, the thickness of both ears was measured. Values represent the mean ± SD of 6 determinations from 3 mice. **P < .01 and ***P < .001; significantly different from positive control.

Oral administration of persimmon leaf extract or astragalin inhibits serum IgE elevation and development of dermatitis in NC/Nga atopic dermatitis model mice 

Subsequently, to examine whether persimmon leaf extract or astragalin might be effective for allergic diseases in vivo, NC/Nga mice were used. NC/Nga mice have been recently recognized as atopic dermatitis–model mice.18, 19 This mouse spontaneously develops severe eczema with aging under nonspecific pathogen free (SPF) circumstances. In addition, the histology of its dermatitis mimics human atopic dermatitis, and the serum IgE level is significantly elevated after the onset of dermatitis. Five mice in each group were orally given persimmon leaf extract (250 mg/kg), astragalin (1.5 mg/kg), or a control diet. Development of dermatitis was observed in the control mice: mild eczema of the head and neck at 10 weeks of age; mild to moderate eczema and hemorrhage of the head and neck, dorsal skin, and ears at 11 weeks; and clear alopecia of the armpit and breast at 13 weeks. However, oral intake of persimmon leaf extract or astragalin remarkably inhibited the appearance of these skin symptoms. Representative skin is shown in Fig 4.

  • View full-size image.
  • Fig. 4. 

    Clinical features of NC/Nga skin. NC/Nga mice were provided with one of the following diets ad libitum: A, control diet (MF diet containing 0.125% of α-corn starch, n = 5); B, persimmon leaf extract diet (MF diet containing 0.125% of persimmon leaf extract at 250 mg/kg, n = 5); or C, astragalin diet (control diet plus astragalin at 1.5 mg/kg, n = 5) during the 8-week experiment. The photograph was taken at 13 weeks.

The skin conditions were evaluated weekly by artificial skin severity score, and the results are shown in Fig 5.
  • View full-size image.
  • Fig. 5. 

    Effect of persimmon leaf extract or astragalin on clinical scores of skin symptoms and serum IgE level. Clinical scores and serum IgE level were measured. Values represent the mean ± SD. *P < .05 and **P < .01; significantly different from control diet.

There was a striking difference in skin severity score between controls and persimmon leaf extract– or astragalin-treated mice. Oral administration of astragalin almost completely suppressed the development of dermatitis and also inhibited scratching behavior (data not shown). The histologic study revealed that in control mice at 13 weeks of age, thickening of the epidermis, prominent hyperkeratosis, and infiltration of inflammatory cells were observed (Fig 6).
  • View full-size image.
  • Fig. 6. 

    Histologic features of skin lesions. Hematoxylin and eosin–stained sections of the ear from control NC/Nga mice (A), persimmon leaf extract mice (B) , or astragalin mice (C) .

Morphologic study or staining showed that these infiltrating cells in the dermis were degranulated mast cells with a substantial number of eosinophils and lymphocytes. These changes were more severe in the outside than the inside of the ear. Oral intake of persimmon leaf extract or astragalin also inhibited thickening of the epidermis, hyperkeratosis, and the infiltration of inflammatory cells in the dermis, as shown in Fig 6. Thus these results indicate that oral intake of persimmon leaf extract or its major flavonoid astragalin inhibits the development of dermatitis in NC/Nga mice. Furthermore, the serum level of IgE was also decreased by persimmon leaf extract or astragalin (Fig 5). Serum level of IgE was measurable after 7 weeks of age and was gradually increased with aging. At any time except for 9 weeks of age in persimmon leaf extract mice, there was a significant difference of IgE between control and other groups of mice. In the 13-week-old NC/Nga mice, serum level of IgE was decreased 5-fold by persimmon leaf extract or astragalin. However, as shown in Table I, other isotypes of immunoglobulins, such as IgM, IgG1, IgG2a, IgG2b, and IgG3, in the sera were not decreased by oral intake of astragalin.
Table I. Serum immunoglobulin concentrations in 13-week-old mice
IgMIgG1IgG2aIgG2bIgG3IgE
Control (μg/mL)842 ± 5323500 ± 17852534 ± 11948636 ± 375234 ± 14.86.0 ± 3.9
Astragalin (μg/mL)750 ± 2533476 ± 21171684 ± 5646028 ± 173030 ± 2.61.22 ± 0.3*

Serum concentrations of Igs from 5 mice were measured by using ELISA. Values are shown as means ± SE. A paired Student t test is used to assess the level of significance of the difference (P < .05).

Oral administration of persimmon leaf extract or astragalin inhibits IL-4 and IL-13 synthesis by spleen T cells 

Next, to identify the mechanisms through which IgE elevation is suppressed by oral intake of astragalin, immunologic analyses were carried out when the mice were 13 weeks old. FACS examination of two mice from each group showed that the total number of spleen cells was similar and that the level of CD3+, CD4+, CD8+, or B220+ cells showed no significant difference between control and astragalin-treated mice (Table II).

Table II. Cell population in the spleens of 13-week-old mice
CD3+CD4+CD8+B220+non-T, non-B
Control (%)20.4 ± 0.713.7 ± 0.98.2 ± 0.364.5 ± 0.313.6 ± 3.5
Astragalin (%)27.6 ± 5.819.5 ± 4.68.8 ± 1.063.1 ± 2.39.3 ± 2.3

Whole spleen cells were stained with appropriate FITC- or phycoerythrin-labeled mAb. Positive cells were determined by using FACS analysis. Data is shown as the mean ± SD from two spleens. There is no significant difference between each group.

Then the capacity of spleen T cells to produce IL-4, IL-13, and IFN-γ was measured after anti-CD3 plus anti-CD28 antibody stimulation. Fig 7 shows that the synthesis of TH2 cell-derived cytokines IL-4 and IL-13 was significantly diminished (P < .05) by oral intake of astragalin but that there was little change in IFN-γ synthesis.
  • View full-size image.
  • Fig. 7. 

    IL-4 and IL-13 synthesis by spleen cells from astragalin or persimmon leaf extract (PLE) mice is suppressed. Spleen cells from mice at 13 weeks of age were stimulated with anti-CD3 plus anti-CD28 antibodies for 2 days. IL-4, IL-13, or IFN-γ in the culture supernatants was measured by ELISA. Values represent the mean ± SD. *P < .05.

Finally, the proliferative response of spleen T cells with anti-CD3 plus anti-CD28 antibodies was studied. Spleen cells were incubated with or without both antibodies for 3 days, and then 3H-thymidine incorporation was measured. No stimulation caused 437 ± 37 cpm from control mice and 111 ± 19 cpm from astragalin-treated mice. The stimulation of spleen cells with anti-CD3 plus anti-CD28 antibodies resulted in 29,173 ± 4492 cpm from control mice and 32,408 ± 2727 cpm from astragalin-treated mice. Thus this suppressive effect of astragalin on IL-4 and IL-13 synthesis was not due to a general inhibition of T cell–proliferative response.

Back to Article Outline

DISCUSSION 

We previously reported that persimmon leaf extract contains antiallergic substances that inhibit histamine release by KU812 cells in response to cross-linkage of FcϵRI and that oral administration of persimmon leaf extract suppressed PCA.14 In this study we demonstrated that the major constituent in the persimmon leaf extract, which is responsible for the inhibition of histamine release, might be astragalin and that oral administration of not only persimmon leaf extract but also astragalin inhibited passive cutaneous anaphylaxis reaction in vivo. In addition, both substances prevented the development of dermatitis and IgE elevation in NC/Nga atopic dermatitis model mice.

It has been reported that botanical materials include several constituents with antiallergic and anti-inflammatory actions.20 Typical examples of such plants are Perilla frutescens , Camellia sinensis , Rubus suavissimus , Artemisia princeps , and Aloe arborescens . Antiallergic substances, such as flavonoids, catechins, and caffeines, contained in these plants have been shown to be responsible for these actions. Catechins are known to suppress histamine release of rat mast cells and to inhibit TNF-α production by LPS-stimulated mouse macrophages.21, 22, 23 (–)-Epigallocatechin, (–)-epicatechin gallate, and (–)-epigallocatechin gallate are the main constituents in oolong tea, green tea, and black tea extracts. These extracts are also known to include caffeines with antiallergic activities.24 However, in persimmon leaf extracts these 3 major catechins ([–]-epigallocatechin, [–]-epicatechin gallate, and [–]-epigallocatechin gallate) and caffeines could not be detected. Persimmon leaves are reported to include 0.15% astragalin and 0.04% isoquercitin from MeOH extract12 and 0.09% astragalin, 0.004% isoquercitin, 0.008% quercetin-3-(2 o-galloyl)-glucoside, and 0.014% kaempferol-3-(2 o-galloyl)-glucoside from Me2CO-H2O (7:3) as flavonoids.11 The present persimmon leaf extract used in our study contained 0.59% astragalin. Because pure astragalin also suppressed histamine release from KU812 cells at a dose equivalent to that in the persimmon leaf extract, it is conceivable that the main constituent that is responsible for this inhibition is astragalin in the persimmon leaf extract. Previous reports indicate that fisetin, kaempferol, quercetin, and myricetin have an inhibitory effect on histamine release from rat peritoneal mast cells.8 The effect of astragalin on the inhibition of histamine release in human blood cells was found to be twice as strong as that of kaempferol (data not shown). In addition, Cheong et al9 examined the structure activity relationship of flavonoids for antiallergic actions through analyses of their inhibitory activity on hexosaminidase release from RBL-2H3 rat mast cells. Apigenin, luteolin, 3,6-dihydroxyflavone, fisetin, kaempferol, quercetin, and myricetin were found to be the flavonoids, which strongly inhibit hexosaminidase release, with an inhibitory concentration of 50% at a level of around 1.8 to 7.5 μmol/L. A comparison of the effects of concentrations of such flavonoids on inhibition with references in the literature suggests that astragalin is one of the strongest inhibitors among flavonoids.

To determine whether persimmon leaf extract and astragalin have antiallergic effects in vivo, ddY and NC/Nga mice were used. Oral administration of each material strongly inhibited the PCA reaction. In NC/Nga mice spontaneous development of dermatitis and IgE elevation under non-SPF circumstances are observed.18, 19 Furthermore, histologic analyses have shown that their skin lesions mimic those of human atopic dermatitis. The administration of persimmon leaf extract by mouth led to a striking inhibition of skin symptoms and IgE elevation. Because astragalin showed a similar suppressive effect on dermatitis and on IgE elevation in vivo, these results support the finding that a major antiallergic substance in persimmon leaf extract is astragalin, although it cannot be completely denied that other components in persimmon leaf extract may additively contribute to the effect. Kaempferol and astragalin from the white petals of I balsamina L have been shown to have an antipruritic effect on dextran T40-evoked scratching behavior in mice.13 As for IgE suppression by botanical materials in vivo, only P frutescens extract has been demonstrated to inhibit anti-DNP IgE production in mice immunized with DNP-ovalbumin in alum adjuvant.25

The clinical efficacy of persimmon leaf extract and astragalin on the inhibition of dermatitis may have been mediated through their inhibitory effect on histamine release by FcϵRI+ cells because the histologic study revealed that there were few degranulated mast cells in the dermis of mice who were given astragalin. Furthermore, these materials had an inhibitory effect on IgE synthesis in vivo. It has been shown that IgE synthesis by B cells is primarily regulated by cytokines; TH2-derived cytokines, including IL-4 and IL-13; IgE class-switching factors, upregulated IgE synthesis; and TH1-derived cytokines, especially IFN-γ, are strong inhibitors of IgE synthesis.26, 27, 28 In addition to TH2 cells, basophils, mast cells, and NK1.1T cells have been shown to be IL-4 producers,29, 30 and in fact peripheral blood basophils and tissue localized mast cells were found to produce IL-4.31, 32, 33 We found that oral intake of astragalin or persimmon leaf extract suppressed IL-4 and IL-13 production, but not IFN-γ synthesis, by spleen T cells in response to anti-CD3 plus anti-CD28 antibodies. These changes in the cytokine profile might contribute to IgE suppression. Whether astragalin directly may affect the differentiation of T cells into TH2 cells remains to be determined. We would like to point out here that preliminary experiments revealed that astragalin at the dose of 10 to 30 μmol/L did not inhibit IgE synthesis by LPS plus IL-4–stimulated mouse B cells nor did it suppress IL-4 synthesis by spleen non-B, non-T cells in response to plate-bound IgE plus IL-3. However, persimmon leaf extract and a hydroxylated flavonoid of astragalin, kaempferol, which is possibly converted from astragalin in vivo, showed an inhibitory activity of IL-4 synthesis by non-B, non-T cells.

The presence of astragalin in plants is ubiquitous, and intake of this natural constituent may be effective for allergic disease. Persimmon leaf extract contains more astragalin than is found in other materials. We hope that these observations may contribute to the clinical application of these materials as alternative medicines for allergic diseases.

Back to Article Outline

References 

  1. Williams H, Robertson C, Stewart A, Ait-Khaled N, Anabwani G, Anderson R, et al.  Worldwide variations in the prevalence of symptoms of atopic eczema in the international study of asthma and allergies in childhood. J Allergy Clin Immunol. 1999;103:125–138
  2. Cooper KD. Atopic dermatitis: recent trends in pathogenesis and therapy. J Invest Dermatol. 1994;102:128–137
  3. Grewe M, Bruijnzeel-Koomen CA, Schopf E, Thepen T, Langeveld-Wildschut AG, Ruzicka T, et al.  A role for Th1 and Th2 cells in immunopathogenesis of atopic dermatitis. Immunol Today. 1998;19:359–361
  4. Leung DYM. Atopic dermatitis: immunobiology and treatment with immune modulators. Clin Exp Immunol. 1997;107:25–30
  5. Graham-Brown RA. Therapeutics in atopic dermatitis. Adv Dermatol. 1997;13:3–31
  6. Fewtrell CMS, Gomperts BD. Effect of flavone inhibitors of transport ATPases on histamine secretion from rat mast cells. Nature. 1977;265:635–636
  7. Chakravarty N. The role of plasma membrane Ca++-Mg++ activated adenosine triphosphatase of rat mast cells on histamine release. Acta Pharmacol Toxicol. 1980;47:223–235
  8. Amellal M, Bronner C, Briancon F, Haag M, Anton R, Landry Y. Inhibition of mast cell histamine release by flavonoids and biflavonoids. Planta Med. 1985;1:16–20
  9. Cheong H, Ryu SY, Oak MH, Cheon SH, Yoo GS, Kim KM. Studies of structure activity relationship of flavonoids for the anti-allergic actions. Arch Pharm Res. 1998;21:478–480
  10. Pagonis C, Tauber AL, Pavlotsky N, Simons ER. Flavonoid impairment of neutrophil response. Biochem Pharmacol. 1986;35:237–245
  11. Funayama S, Hikino H. Hypotensive principle of Diospyros khaki leaves. Chem Pharm Bull. 1979;27:2865–2868
  12. Kameda K, Takaku T, Okuda H, Kimura Y, Okuda T, Hatano T, et al.  Inhibitory effects of various flavonoids isolated from leaves of persimmon on angiotensin-converting enzyme activity. J Nat Prod. 1987;50:680–683
  13. Ishiguro K, Oku H. Antipruritic effect of flavonol and 1,4-naphthoquinone derivatives from Impatiens balsamina L. Phytother Res. 1997;11:343–347
  14. Kotani M, Fujita A, Tanaka T. Inhibitory effects of persimmon leaf extract on allergic reaction in human basophilic leukemia cells and mice. J Jpn Soc Nutr Food Sci. 1999;52:147–151
  15. Gounni AS, Lamkhioued B, Ochiai K, Tanaka Y, Delaporte E, Capron A, et al.  High-affinity IgE receptor on eosinophils is involved in defense against parasites. Nature. 1994;367:183–186
  16. Kishi K. A new leukemia cell line with Philadelphia chromosome characterized as basophil precursors. Leuk Res. 1985;9:381–390
  17. Inagaki N, Goto S, Nagai H, Koda A. Mouse ear PCA as a model for evaluating antianaphylactic agents. Int Arch Allergy Appl Immunol. 1984;74:91–92
  18. Matsuda H, Watanabe N, Geba GP, Sperl J, Tsudzuki M, Hiroi J, et al.  Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int Immunol. 1997;9:461–466
  19. Matsumoto M, Ra C, Kawamoto K, Sato H, Itakura A, Sawada J, et al.  IgE hyperproduction through enhanced tyrosine phosphorylation of Janus kinase 3 in NC/Nga mice, a model for human atopic dermatitis. J Immunol. 1999;162:1056–1063
  20. Middleton E, Kandaswami C. Effects of flavonoids on immune and inflammatory cell functions. Biochem Parmacol. 1992;43:1167–1179
  21. Kakegawa H, Matsumoto H, Endo K, Satoh T, Nonaka G, Nishioka I, et al.  Inhibitory effects of tannins on hyaluronidase activation and on the degranulation from rat mensentery mast cells. Chem Pharm Bull. 1985;33:5079–5082
  22. Matsuo N, Yamada K, Shoji K, Mori M, Sugano M. Effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells: the structure-inhibitory activity relationship. Allergy. 1997;52:58–64
  23. Kar K, Mohanta PK, Popli SP, Dhawan BN. Inhibition of passive cutaneous anaphylaxis by compounds of Camellia sinensis. Planta Medica. 1981;42:75–78
  24. Ohmori Y, Ito M, Kishi M, Mizutani H, Katada T. Antiallergic constituents from oolong tea stem. Biol Pharm Bull. 1995;18:683–686
  25. Foreman JC. Mast cells and the actions of flavonoids. J Allergy Clin Immunol. 1984;73:769–774
  26. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986;136:2348–2357
  27. Paul WE, Seder RA. Lymphokine responses and cytokines. Cell. 1994;76:241–251
  28. Romagnani S. Lymphokine production by human T cells in disease states. Annu Rev Immunol. 1994;12:227–257
  29. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol. 1994;12:635–673
  30. Yoshimoto T, Paul WE. CD4+NK1.1+T cells promptly produce interleukin-4 in response to in vitro challenge with anti-CD3. J Exp Med. 1994;179:1285–1295
  31. Ochi H, Tanaka T, Katada Y, Naka T, Aitani M, Hashimoto S, et al.  Peripheral blood T lymphocytes and basophils, freshly isolated from house dust mite sensitive patients, produce interleukin-4 in response to allergen-specific stimulation. Int Arch Allergy Immunol. 1996;111:253–261
  32. Bradding P, Feather IH, Howarth PH, Mueller R, Roberts JA, Britten K, et al.  Interleukin 4 is localized to and released by human mast cells. J Exp Med. 1992;176:1381–1386
  33. Horsmanheimo L, Harvima IT, Jarvikallio A, Harvima RJ, Naukkarinen A, Horsmanheimo M, et al.  Mast cells are one major source of interleukin-4 in atopic dermatitis. Br J Dermatol. 1994;131:348–353

☆ Supported in part by the Ministry of Education, Science, and Culture of Japan; the Ministry of Health and Welfare of Japan; Astra Research Grant for Asthma Research; Japan Research Foundation for Clinical Pharmacology; and the Pollution-related Health Damage Compensation and Prevention Association of Japan.

☆☆ Reprint requests: Toshio Tanaka, MD, PhD, Department III of Internal Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan.

PII: S0091-6749(00)90951-5

doi:10.1067/mai.2000.107194

The Journal of Allergy and Clinical Immunology
Volume 106, Issue 1 , Pages 159-166, July 2000

Article Tools

* Image Usage & Resolution