WO2010041831A2 - Anti-allergy active composition containing a compound derived from ecklonia cava - Google Patents

Abstract

The present invention relates to a composition having an anti-allergy activity, containing a phlorotannin compound separated from the extract of Ecklonia cava. The Ecklonia cava-derived phlorotannin compound according to the present invention is useful for preventing allergic reactions caused by allergen-IgE, multifunctionally hinders the release of histamine, and inhibits the binding of IgE and the FceRI receptor to exhibit excellent anti-allergy activity.

Description

Anti-allergic active compositions containing compounds derived from Ecklonia cava

The present invention relates to an anti-allergic active composition containing an Ecklonia cava- derived compound, and to a food, cosmetic or pharmaceutical composition having an anti-allergic activity containing a phlorotannin compound isolated from Ecklonia cava as an active ingredient. will be.

Today, approximately one third of the total population suffers from allergic diseases. Allergic diseases refer to pathological phenomena due to antigen-antibody reactions. Serum diseases, high fever, and bronchial asthma are typical. Nowadays, however, there is a tendency to consider it as a disease that can be caused by allergies, which is very similar to the disease in which the antigen-antibody reaction is proved, but that the antigen-antibody reaction is not proven, or the involvement of the antigen-antibody reaction is not proved, but it is highly It also includes things. In other words, it can be regarded as an idea based on reality, and common to these include genetic or constitution factors, common causes, and commonality of symptoms and drug effects.

Examples of allergic diseases include organs such as bronchial asthma, hyperthermia, vasomotor neuritis, hypertrophic rhinitis, allergic bronchitis, Loupler syndrome (transient pulmonary infiltration), etc. Diarrhea, allergic stomatitis, entero purpura (腸 性 紫斑病), circulatory system, such as nodular periarthritis, crystalline endocarditis, angina pectoris, endocarditis, etc. The eye and the like are flictene, sympathetic ophthalmitis, allergic conjunctivitis, allergic keratitis and the like, and other examples include diffuse glomerulonephritis and migraine headaches.

In addition, allergic diseases are classified into immediate type (卽 時 型: anaphylaxis type) and late type (遲發型: tuberculin type). Immediate forms include high fever, bronchial asthma, urticaria, quinque edema, allergic digestive tract, etc., and delayed forms include contact dermatitis and drug allergies. There is some agreement on the therapeutic effect of the reactor and the drug.

Allergic diseases include a wide range of other inflammatory mediators such as eicosanoids, proteoglycans, proteases, as well as endogenous mediators such as histamine, and several such as tumor necrosis factor (TNF), interleukin (IL-6, IL-4, IL-13). It is caused by chemical or immunological activation of mast cells resulting in excessive release of proinflammatory and chemotactic cytokines. Of the inflammatory substances released from effector cells, histamine is the most characterized and potent vascular mediator involved in the acute hypersensitivity of immediate hypersensitivity and is considered a marker of degranulation. Inhibition of degranulation from these cells is one of the important steps in the prevention of allergic diseases. The search for novel substances that can prevent them in the early stages of allergic activity is an essential strategy in the treatment of atopic diseases.

Among natural sources, marine algae, which is widely consumed as a food in many Asian countries, especially in Korea and Japan, can be a very useful source for therapeutic purposes. Many of these have been reported to have various biological activities, such as anticancer, antioxidant and anti-allergic activities. In recent years, brown alga-derived phloroglucinol derivatives have received much attention due to their broad therapeutic prospects.

These phlorotannins have been shown to exhibit a variety of biological activities including antidiabetic, antioxidant, anticancer, anti-inflammatory and anti-HIV. Interestingly, several reports have been published on the anti-allergic activity of phloroglucinol compounds. Phlorotannin showed a high inhibitory effect on RBL 2H3.

For example, Korean Patent Application No. 10-2006-7009567 "Atopic Dermatitis, Skin Allergy Symptoms and Acne Therapeutic Composition" includes a) of Ginkgo biloba for the treatment of atopic dermatitis, skin allergy symptoms and acne. Terpenes; b) floroglucinols extracted from Humulus lupulus, Hypericum sp and Mirtus sp, pure or mixtures thereof; c) A topical composition is disclosed which comprises ageotic extract of Zanthoxylum bungaenum or Echinacea angustifolia.

Ecklonia cava Eye edible brown algae, widely distributed in the southern coast of Korea and Japan. They are produced abundantly in Jeju Island, Korea (30,000 tons / year), but are not used in Europe. These valuable brown algae are used as folk medicines in foodstuffs, animal feed, fertilizers and gynecological diseases in Korea and Japan.

Specifically, ecstasy is mainly used in food or cosmetics, for example, a cosmetic composition for whitening (Korean Patent Application No. 10-2000-0048933), a cosmetic composition for improving wrinkles (Korean Patent Application No. 10-2001-0025580), acne It is used as an active ingredient of a cosmetic composition for skin (Korean Patent Application No. 10-2006-0093939).

In addition, regarding the use of phloroglucinol compounds derived from Ecklonia cava, Korean patent application No. 10-2002-83555 "degenerative arthritis composition comprising seaweed extract" for the prevention and improvement of osteoarthritis, including Ecklonia Disclosed is a degenerative arthritis composition comprising seaweed extract containing more than 10% polyfluoroglucinol complex consisting of 15 polyphenols extracted from the seaweed, Korean Patent Application No. 10-2004-3912 "cardiovascular disease Improved composition "is disclosed a cardiovascular disease improving composition comprising alginic acid, fucoidan, laminarin, phloroglucinol and fucosterol derived from algae comprising Ecklonia.

In addition, Korean Patent No. 10-0518179, "Performance Improvement Composition," is a powerful antioxidant that protects vascular endothelial cells from free radicals to maintain the relaxation state of vascular muscle, which is the core of erectile maintenance, and vascular contraction. Algae extract powder containing Ecklonia chinensis which contains essential fucosterol ingredients that regulate the activity of ACE (ANGIOTENSIN CONVERTING ENZYME), which is known to interfere with erections and various plant raw materials that provide essential nutrients for energy maintenance and sexual function A sexual function improving composition is disclosed which has excellent effects on improving sexual function without any side effects.

The present inventors have conducted extensive studies to separate anti-allergic activity from natural substances, particularly seaweeds, and have purified methanol extracts from Ecklonia cava to continually identify the components that cause anti-allergic effects. After purifying a single active ingredient from the ethyl acetate fraction which showed the highest activity, their anti-alled group activity was confirmed to arrive at the present invention.

Therefore, an object of the present invention is to isolate and purify the phlorotannin compound having various physiological activities from Ecklonia cava , which is a brown seaweed.

It is also an object of the present invention to provide a novel anti-allergic activity of the chlorotannin compound derived from Ecklonia cava .

The object of the present invention as described above is to purify the methanol extract derived from Ecklonia cava, to isolate a single component from the ethyl acetate fraction showing the highest activity, and to reveal the structure of the four phlorotannin compounds according to NMR data, respectively, Degranulation levels visualized histamine and β-hexosaminidase release to basophil leukemia (KU812F) cell lines and rat basophil leukemia (RBL-2H3) cell lines were measured to reveal their anti-allergic activity.

The present invention provides an anti-allergic active food composition containing an Ecklonia cava- derived phlorotannin compound as an active ingredient.

The present invention also provides an anti-allergic active cosmetic composition containing as an active ingredient an ectopically derived phlorotannin compound.

The present invention provides an anti-allergic active pharmaceutical composition containing an ectopically derived phlorotannin compound as an active ingredient.

The phlorogannin compound of the present invention is phloroglucinol, diexol, 6,6′-bieckol or 1- (3 ′, 5′-dihydro-xyphenoxy) -7- of Formulas 1 to 4 (2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1,4-dioxin [1- (3 ', 5'-dihydro-xyphenoxy) -7 -(2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1,4-dioxin].

<Formula 1>

Floroglucinol

<Formula 2>

                  Dieckol

<Formula 3>

             6,6’-bieckol

<Formula 4>

1- (3 ', 5'-Dihydro-Zyphenoxy) -7- (2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1,4 Dioxin

In the present invention, the food composition as an active ingredient of the Ecklonia cava-derived phlorotannin compound according to the present invention may be added to impart anti-allergic activity to dietary supplements, special nutritional products, beverages and alcoholic beverages, food additives, confectionery or bread. However, the present invention is not limited thereto, and may be used for various other foods.

In the present invention, the cosmetic composition containing the Ecklonia cava derived pharotannin compound according to the present invention as an active ingredient may vary depending on the type of cosmetic. Cosmetic composition according to the present invention means a cosmetic composition that exhibits anti-allergic activity, including an ectopically derived phlorotannin compound, and may be in various forms, for example, in liquid, cream, paste or solid form. However, the present invention is not limited thereto and may be used in various other forms of cosmetics.

In the present invention, as an allergic disease to which the anti-allergic active pharmaceutical composition containing the Ecklonia harmful phlorotannin compound according to the present invention as an active ingredient, as an respiratory disease, bronchial asthma, high fever, blood vessels Diseases of the digestive system such as motorized rhinitis, hypertrophic rhinitis, allergic bronchitis, Loepller syndrome (transient lung infiltration), dietary allergic gastritis, allergic diarrhea, allergic stomatitis, entero purpura, circulatory system Diseases such as nodular periarteritis, obstructive arteritis, angina pectoris, endocarditis, urticaria, quinque edema (vascular neuroedema), nodular erythema, purpura, and fever, sympathetic ophthalmitis, allergic conjunctivitis , Allergic keratitis, and the like include diffuse glomerulonephritis and migraine headaches. .

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the drug directly or indirectly by inherent pharmacological action. It means containing a main component).

The ectopically derived phlorotannin compound according to the present invention showed low cytotoxicity against the test cell line. At 500 μM concentration, the lowest cell viability of KU812 and RBL-2H3 was 91.76% (fluoroglucinol, compound 1) and 93.66% (dieckol, compound 2), respectively. In other treatments, the compounds showed no significant cytotoxic effect on the test cell line.

Relative histamine release levels for antibody-promoted KU812 cells at the highest concentrations of Compounds 1-4 were 93.22%, 11.99%, 12.08%, 74.54%, respectively, and relative histamine release rate for KU812 cells promoted by A23187. 102.36%, 40.05%, 39.49%, 69.08%, respectively, and the relative histamine release rates for RBL-2H3 cells were 98.0296%, 62.68%, 50.64%, 54.98%, and relative β-hexosamini for RBL-2H3 cells, respectively. Aze release rates were 90.37%, 35.26%, 18.66% and 49.36%, respectively.

The phlorotannin compound according to the present invention showed a potent and dose dependent inhibitory effect on histamine released from cell lines and stimulants. From this, the phlorotannin compound according to the present invention may be useful for preventing allergen-IgE-induced allergic reactions, and has an effect of multifunctionally inhibiting hydramine release.

The phlorotannin compounds according to the invention can also inhibit the histamine release promoted by A23187 in each cell line by mitigating an increase in intracellular Ca ²⁺ levels corresponding to a decrease in histamine release from the cells by stabilizing the cell membrane. there was.

In addition, the phlorotannin compound according to the present invention was found to be able to inhibit binding between IgE and FcεRI receptor in a dose-dependent manner through flow cytometry.

The chlorophyllin compound derived from Ecklonia cava according to the present invention is useful for preventing allergen-IgE-induced allergic reaction, can multiply inhibit hydramine release, and can inhibit the binding between IgE and FcεRI receptor. Allergic activity has a very good effect.

1 is a diagram showing the chemical formula of an ectopically derived phlorotannin compound.

FIG. 2 shows cytotoxicity levels of phlorotannin compounds against human basophilic leukemia (KU812F) cell lines analyzed by MTT cell viability assay at different concentrations.

FIG. 3 shows cytotoxicity levels of phlorotannin compounds against rat basophilic leukemia (RBL-2H3) cell lines analyzed by MTT cell viability assay at different concentrations.

4 is a diagram showing the effect of phlorotannin compound on FcεRI activated histamine release rate in KU812 cells.

5 is a diagram showing the effect of phlorotannin compound on the release rate of A23187 activated histamine in KU812 cells.

FIG. 6 shows the effect of phlorotannin compounds on FcεRI activated histamine release rate in RBL-2H3 cells. FIG.

Figure 7 shows the effect of phlorotannin compound on A23187 activated histamine release rate in RBL-2H3 cells.

8 to 10 are diagrams showing the effect of phlorotannin compounds on the binding activity between IgE and FcεRI on the surface of KU812 cells.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example

In this example, ¹ H NMR (400 MHz) and ¹³ C NMR (100 MHz) spectra are JEOL JNMECP using DMSO- d 6 solvent peak (d 2.50 ppm in 1H and d 39.5 ppm in 13C NMR) as internal reference standard. It was recorded with a 400 NMR spectrometer (JEOL, Japan). For some signals, chemical shifts were recorded to three decimal places. This is to distinguish signals that are very close in value but can still be clearly identified by visual inspection of the spectrum.

MS spectra were obtained on a JEOL JMS-700 spectrometer (JEOL, Japan). Extraction of EC was performed using an extraction unit (Dongwon Scientific Co., Korea). Column chromatography was performed with silica gel 60 (230-400 mesh, Merck Germany), Sephadex LH-20 (Sigma, st. Louis, MO, USA). TLC was run on a pre-coated Merck Kieselgel 60 F254 plate (0.25 mm), and the points on the TLC plate were converted into CHCl ₃ : MeOH: H ₂ O: acetic acid (65: 25: 4: 3, v / v) using a UV lamp (254 & 365 nm) and vanillin-H ₂ SO ₄ was used as a detection agent for phenolic acid compounds. All solvents for column chromatography used reagent grades obtained from commercial sources.

Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 medium, trypsin-EDTA, penicillin / streptomycin / amphotericin (10,000 U / mL, 10,000 μg / mL, and 2500 μg / mL) and right Fetal bovine serum (FBS) was obtained from Gibco BRL Light Life Technologies (USA). Human myeloma IgE was purchased from Calbiochem (Gibbbstown, NJ 08027, USA). Goat anti-human IgE was purchased from Gentex (San Antonio, TX, USA). Let anti-DNP monoclonal antibody, unconjugated IgE, was purchased from Invitrogen (Carlsbad, Calif., USA). Fluorescence isothiocynate (FITC) -conjugated anti-human IgE antibodies were purchased from Biosources (Burlingame, CA, U.S.A). All other reagents such as Calcium inonophore (A23187), hydroxyethyl piperazinylethanesulfonic acid (HEPES), L-glutamine histamine, histamine, dimethyl sulfoxide (DMSO) and Ortho-phthalaldehyde (OPA) was purchased from Sigma Chemicals (St. Louis, MO, USA). Other chemicals and solvents used those of analytical grade.

Statistical analysis of all experimental results was expressed as mean ± standard deviation ( n = 3). The significance level was determined using the Student's t test.

Example 1 Extraction and Separation of Phlorotannin Compounds from Ecklonia cava

Example 1-1 Preparation of Ecklonia cava Extract and Isolation of Phlorotannin Compound

Ecklonia cava (EC), a marine edible brown seaweed, was collected from Jeju Island, Korea, from October 2004 to May 2005. Fresh EC was washed three times with water to remove salt. Lyophilized EC was powdered and used for extraction. The dried EC powder (10 kg) was extracted by stirring extraction with MeOH (3 × 5 L) for 10 days.

The extract (273 g) was suspended in water and partitioned successively with n -hexane (35.92 g), CH ₂ Cl ₂ (20.49 g), EtOAc (24.87 g), n- BuOH (106 g). Ten subfractions (fractions 1 to 10) were obtained by silica gel flash chromatography eluting with EtOAc / EtOAc / MeOH (gradient), EtOAc fraction (24.87 g) showing the strongest anti-allergic activity against human cells. Fraction 5 (378.39 mg) having the highest activity against anti-allergic groups was further purified with Sephadex LH-20 using MeOH to give Compound 1 (102.85 mg), Compound 2 (58.30 mg), compound 3 (47.62 mg), and compound 4 (53.71 mg) were obtained.

Example 1-2 Structural Analysis of Florotannin Compounds

In this example, the structures of the phlorotannin compounds 1 to 4 isolated in Example 1-1 were analyzed.

Compound 1 was found to have molecular formula C ₆ H ₆ O ₃ as measured by EIMS, ¹ H, ¹³ C NMR, and DEPT spectral data. In the NMR spectrum of Compound 1, three phenolic acid OH protons at d 8.97 (3H, s) and three aromatic singlet resonances at d 5.66 (3H, s) were observed. ¹³ C and DEPT NMR spectra showed two peaks at 94.1 (d) and 158.9 (s). In addition to MS data ( m / z 126), Compound 1 was clearly identified as fluorologusinol as shown in Formula 1 of FIG.

Compound 2 was obtained as a pale brown powder and the molecular formula was determined as C ₃₆ H ₂₂ O ₁₈ according to EIMS, ¹ H, ¹³ C NMR, and DEPT spectral data. ¹ H NMR spectra excited the AB ₂ system at d 5.80 (1H, t, J = 2.0 Hz, H-4 ′, 5.78 (2H, d, J = 2.0 Hz, H-2 ′, 6 ′), d 5.82 (1H, d, J = 2.7 Hz, H-6), 6.02 (1H, d, J = 2.7 Hz, H-8) and 5.98 (1H, d, J = 2.7 Hz, H-8 "), 5.81 ( 1H, d, J = 2.7 Hz, H-6 ") resulting in two AB ₂ systems. D 6.17 (1H, s, H-3"), 6.14 (1H, s, H-3), 5.95 ( D 9.71 (1H, s, OH-9), 9.61 (1H, s, OH-9 "), 9.51 (1H, s) as well as three singlets at 1H, s, H-2"', 6 "' , OH-4 "), 9.46 (1H, s, OH-4), 9.36 (2H, s, OH-3", 5 "), 9.28 (1H, s, OH-2"), 9.23 (1H, s , OH-2), 9.22 (1H, s, OH-7 ") and 9.15 (2H, s, OH-3 ', 5', 11 phenolic protons were resonated. The ¹³ C NMR spectrum shows compound divalent 9 It indicates that it contains 2 unsubstituted and 23 O-containing aromatic carbons.By comprehensive analysis of these data and comparison with existing data, Compound 2 was determined to be dieckol, such as Formula 2 of FIG. .

Compound 3 was obtained as a pale brown amorphous powder. The molecular formula of compound 3 was determined as C ₃₆ H ₂₆ O ₁₈ according to EIMS, ¹ H, ¹³ C NMR, and DEPT spectral data. The ¹ H NMR spectra of Compound 3 were obtained from two AB2 systems at d 5.75 (2H, J = 2.2 Hz), 5.80 (1H, J = 2.2 Hz), and at d 6.05 (2H, s) and 6.09 (2H, s). 12 phenolic hydroxyl protons at d 9.29 (2H, s), 9.16 (4H, s), 9.15 (2H, s), 9.09 (2H, s), 8.65 (2H, s) as well as two singlet signals Indicated. ¹³ C NMR spectra showed the presence of two quaternary aromatic carbon signals as well as 10 unsaturated and 12 O-containing aromatic hydrocarbons. The molecular weight of compound 3 was twice that of Exol due to only two unit differences (746 vs. 372). This data indicated that Compound 3 consisted of six phloroglucinol units. According to the data and the comparison with the conventional literature, Compound 3 was determined by 6,6'-bieckol as shown in Formula 3 of FIG.

Compound 4 was separated into a pale brown powder and its molecular formula was determined to be C ₂₄ H ₁₈ O ₁₂ according to EIMS, ¹ H, ¹³ C NMR, and DEPT spectral data. ¹ H NMR spectra were obtained from the AB2 system at d 5.71 (2H, J = 1.8 Hz), 5.79 (1H, t, J = 1.8 Hz), d 5.77 (1H, d, J = 2.6 Hz), 6.00 (1H, d, J system at 2.6 Hz) and two singlets at 6.14 (1H, s) and 5.85 (2H, s), d 9.65 (1H, s), 9.45 (1H, s), 9.24 (1H, s) , 8 phenolic resonances at 9.16 (2H, s), 9.15 (2H, s) and 9.03 (1H, s). ^{In the 13} C NMR spectrum, 8 unsubstituted and 16 O-containing aromatic carbons were observed. The above data for compound 4 are in agreement with previously known data. It is completely 1- (3 ', 5'-dihydro-zaphenoxy) -7- (2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo- 1,4-dioxin [1- (3 ', 5'-dihydro-xyphenoxy) -7- (2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1,4-dioxin] Turned out.

Compound 1: off-white powder; 1H NMR (DMSO- d 6, 400 MHz) d 8.97 (3H, s, OH-1, 3, 5), 5.66 (3H, s, H-2, 4, 5); 13C NMR (DMSO- d 6, 100 MHz) d 94.1 (C-2, 4, 6), 158.9 (C-1, 3, 5); LREIMS m / z 126.05 [M] &lt; + &gt;.

Compound 2: light brown powder (lyophilized), 1 H NMR (DMSO- d 6, 400 MHz) d 9.71 (1H, s, OH-9), 9.61 (1H, s, OH-9 ', 9.51 (1H, s, OH-4 "), 9.46 (1H, s, OH-4), 9.36 (2H, s, OH-3", 5 "), 9.28 (1H, s, OH-2"), 9.23 (1H, s, OH-2), 9.22 (1H, s, OH-7 "), 9.15 (2H, s, OH-3 ', 5', 6.17 (1H, s, H-3"), 6.14 (1H, s , H-3), 6.02 (1H, d, J = 2.7 Hz, H-8), 5.98 (1H, d, J = 2.7 Hz, H-8 "), 5.95 (1H, s, H-2"' , 6 "', 5.82 (1H, d, J = 2.7 Hz, H-6), 5.81 (1H, d, J = 2.7 Hz, H-6"), 5.80 (1H, t, J = 2.0 Hz, H -4 "), 5.78 (2H, d, J = 2.0 Hz, H-2 ', 6'; 13C NMR data (see Table 1), LREIMS m / z 743.10 [M] +.

Compound 3: light brown powder (lyophilized), 1 H NMR (DMSO- d 6, 400 MHz) d 9.29 (1H, s, OH-9), 9.16 (2H, s, OH-3 ', 5', 9.15 (1H, s, OH-2), 9.09 (1H, s, OH-4), 8.65 (1H, s, OH-7), 6.09 (1H, s, H-3), 6.05 (1H, s, H -8), 5.80 (1H, d, J = 2.2 Hz, H-4 ', 5.75 (2H, d, J = 2.2 Hz, H-2', 6 ', 13C NMR data (see Table 1); LREIMS m / z 743.12 [M] &lt; + &gt;.

Compound 4: pale brown powder (lyophilized), 1H NMR (DMSO- d 6, 400 MHz) d 9.65 (1H, s, OH-9), 9.45 (1H, s, OH-4), 9.24 (1H, s, OH-2), 9.16 (2H, s, OH-3 ', 5', 9.15 (2H, s, OH-2 ", 6"), 9.03 (1H, s, OH-4 "), 6.14 ( 1H, s, H-3), 5.77 (1H, d, J = 2.6 Hz, H-6), 6.00 (1H, d, J = 2.6 Hz, H-8), 5.85 (2H, s, H-3 ", 5"), 5.71 (2H, J = 1.8 Hz, H-2 ', 6', 5.79 (1H, t, J = 1.8 Hz, H-4 '; 13C NMR data (see Table 1); LREIMS m / z 496.13 [M] &lt; + &gt;.

TABLE 1

¹³ C NMR data for phlorotannin (compounds 2, 3 and 4) in DMSO- _d6

a: recorded in ¹³ C and ¹³ C DEPT 100 MHz NMR

b: dieckol, c: 6,6'-bieckol,

d: 1- (3 ', 5'-dihydro-zaphenoxy) -7- (2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1 , 4-dioxin

Example 2 Analysis of Biological Activity of Florotannin Compounds

Example 2-1: Cell Culture

RBL-2H3 in human leukemia cell line KU812 and rat basophil leukemia cells was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) .Ku812 and RBL-2H3 were maintained in RPMI-1640 medium and DMEM medium, respectively. Supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES buffer, 100U / mL penicillin G, 100 mg / mL streptomycin, and incubated in a wet environment with 5% CO ₂ at 37 , Subculture every 3-4 days.

Example 2-2 Analysis of Cytotoxicity Levels of Phlorotannin Compounds on Test Cells

Cytotoxic levels of phlorotannin compounds against KU812 and RBL-2H3 cells were measured using the MTT (3- (4,5-dimethyl-2-yl) -2,5-diphenyltetrazolium bromide) method. Cells were incubated at 1 × 10 ⁵ cells / well density in 48-well plates. After 24 hours, cells were washed with fresh medium and treated with different concentrations of phlorotannin compounds. After 48 hours of incubation, the cells were rewashed, 40 μL of MTT (5 mg / mL) was added and then incubated for 4 hours. The amount of formazan salt was finally determined by dissolving the formazan salt formed by adding DMSO (250 μL) and measuring the OD at 540 nm using a GENios microplate reader (Tecan Austria GmbH, Austria). Relative cell viability was measured by the amount of MTT converted to formazan salt. The viability of the cells was quantified in% compared to the control (OD of the treated cells OD / OD of the OD-blank of control) and a dose-dependent curve was created. Data is presented as mean from three or more trials, with P <0.05 considered significant.

As described above, before testing the anti-allergic effect of phlorotannin compounds on two cell lines (KU812 and RBL-2H3), their cytotoxic levels were analyzed using the MTT assay. As shown in Figure 2, the concentration of the test compound was used 5 times higher than in the experimental example. The phlorotannin compound showed low cytotoxicity against the test cell line. At 500 μM concentration, the lowest cell viability of KU812 and RBL-2H3 was 91.76% (Compound 1) and 93.66% (Compound 2), respectively. In other treatments, the compounds showed no significant cytotoxic effect on the test cell line.

Example 2-3 Histamine Release Assay and Histamine Measurement

KU812 cells (5 × 10 6 cells / mL) were preincubated with human IgE (10 μg / mL) for 1 hour and incubated with samples of different concentrations after 30 minutes. The mixture was then incubated for 30 minutes with goat anti-human IgE. Supernatants were collected for histamine measurements. KU812 and RBL-2H3 cells were incubated with 2 μM of stimulant for 30 minutes, then treated with different concentrations of samples and the supernatants collected for histamine measurements.

The histamine content was measured by a slightly modified fluorometric assay. KU812 cells (5 × 10 ⁶ cells / mL) were treated with different concentrations of phlorotannin compound for 30 minutes. The treated cells were resuspended and stimulated with antibody or A23187 and the supernatants collected. After centrifugation, 1 N NaOH and 0.2% OPA were added and incubated for 5 minutes. 3 N HCl was added to terminate the reaction. Fluorescence intensity was measured via excitation wavelength at 365 nm and emission wavelength at 465 nm. The histamine release rate (%) was calculated by the following formula.

% Histamine release rate = (test-negative control) / (positive control-negative control) X 100.

The supernatant from unstimulated cells was used as a negative control, and the supernatant from stimulating cells using anti-FcεRI antibody was used as a positive control.

1) Analysis of the effect of phlorotannin on antibody-promoted KU812 cell histidine release

In this example, the inhibitory effect of the compounds on degranulation of KU812 cells promoted by IgE and anti-IgE antibodies was analyzed. KU812 cells (1 × 10 ⁶ cells / mL) were pre-incubated with human IgE (10 μg / mL) for 1 hour and then incubated with different concentrations of samples for 30 minutes. The mixture was then incubated with goat antihuman IgE for 30 minutes. The histamine content in the supernatant was measured by the fluorometic method.

As shown in FIG. 2B, the relative histamine release rate levels for KU812 cells at the highest concentrations of Compounds 1-4 were 93.22%, 11.99%, 12.08%, 74.54%, respectively.

2) Effect of phlorotannin on KU812 cell histamine release stimulated by A23187

In this example, the inhibitory effects of compounds on the degranulation of KU812 cells mediated by calcium ion permeate carrier (A23187) were analyzed. KU812 cells were incubated for 30 minutes with 2 μM A23187 and then treated with different concentrations of samples. The histamine content in the supernatants was measured by flow cytometry. As shown in FIG. 3A, the relative histamine release rates for KU812 cells at the highest concentrations of Compounds 1-4 were 102.36%, 40.05%, 39.49%, 69.08%, respectively.

3) Effect of phlorotannin on RBL-2H3 cell histamine release promoted by A23187

In this example, the inhibitory effect of the compounds on the degranulation of RBL-2H3 cells mediated by calcium ion permeate carrier (A23187) was analyzed. RBL-2H3 cells were incubated for 30 minutes with 2 μM A23187 and then treated with different concentrations of samples. The histamine content in the supernatants was measured by flow cytometry. As shown in FIG. 3B, the relative histamine release rates for RBL-2H3 cells at the highest concentrations of Compounds 1-4 were 98.0296%, 62.68%, 50.64%, 54.98%, respectively.

Example 2-3 RBL-2H3 Cell Stimulation and β-Hexosaminidase Release Assay

The release of mast cell mediators by extracellular flux was monitored using a conventional β-hexosaminidase assay with a slight modification. RBL-2H3 cells were plated at ⁵ × 10 ⁵ cells / 400 μL / well in 24-well plates and incubated overnight with anti-DNP IgE (0.5 μg / mL, final concentration). The supernatant was decanted and the cells washed three times with release buffer (25 mM Pipes, pH 7.2, 125 mM NaCl, 2.7 mM KC1, 5.6 mM glucose, 1 mM CaCl2, and 0.1% BSA). The cells were then incubated in the presence of varying concentrations of test compound for 37 to 15 minutes. Then they were stimulated for 20 minutes by adding 10 ng / mL DNP-BSA. An aliquot (10 μL) of the cell eluate obtained by adding 200 μL of the medium and Triton X-100 was added to 10 μL of 1 M p -nitrophenyl- N -acetyl-β-d-glucosamide in 0.1 M sodium citrate (pH 4.5). Incubated at 37 for 1 hour. After the incubation was completed, 250 μL of carbonate buffer containing 0.1 M, Na 2 CO 3 and 0.1 M NaHCO 3 (pH 10) was added, and the absorbance attributable to the formation of p -nitrophenol was measured at 405 nm. The total release rate of β-hexosaminidase was calculated by the following formula.

Total Release Rate (%) = [( A - C ) / ( B - C ) ] X 100

Wherein A is the amount of β-hexosaminidase in the extracellular fluid,

B is the total amount of β-hexosaminidase in the cell,

C is the amount of β-hexosaminidase in the non-stimulatory cell derived extracellular fluid.

% Inhibition = [1- (total release rate (%) / stimulated cell release rate (%) accompanying the test compound]] X 100

As described above, to analyze the inhibitory effect of the compound on degranulation of RBL-2H3 cells mediated by IgE promotion via granulation enzyme β-hexosaminidase, 0.5 μg / mL of RBL-2H3 cells were measured. After incubation with IgE antibody for 1 hour, the samples were treated with an upper concentration sample. DNP-BSA was added to the plates to cause degranulation of the cells. As shown in FIG. 3C, the relative β-hexosaminidase release rates for RBL-2H3 cells at the highest concentrations of Compounds 1-4 were 90.37%, 35.26%, 18.66%, 49.36%, respectively.

Example 2-4 Flow Cytometry of Binding Activity Between Human IgE and FcεRI Receptors on KU812F Cells

The binding activity between human IgE and FcεRI receptors on KU812F cells was analyzed by indirect immunofluorescence flow cytometric method. Briefly, KU812F cells (1 × 10 ⁶ cells / mL) were incubated with human IgE antibody (10 μg / mL). Cells were then washed with ice cold PBS and incubated with FITC-conjugated anti-human IgE antibody (2.5 μg / mL). After washing with ice cold PBS, the cells were suspended in 1 mL PBS and treated with a flow cytometer (Beckman Coulter Epics XL, USA) having a wavelength range of 475-525 nm. % Positive cells were calculated with any 1% cutoff channel position measured by the negative control.

As a result of analyzing the binding activity between IgE and FcεRI receptor on KU812 cells by indirect fluorescent flow cytometry in this Example, Compound 1 showed almost no inhibitory effect on degranulation as well as binding. As shown in FIG. 4A, Compound 2 showed the strongest inhibitory effect (85%, see FIG. 4A) at the highest concentration, followed by Compound 4 (78%, see FIG. 4C), and Compound 3 compared to the blank treatment. (63%, see FIG. 4B). The proportion of positive cells in the staining pattern was calculated by arbitrary 1% cutoff channel position determine for the negative control.

Chemical mediators in acute allergic reactions cause a variety of pathophysiological consequences, for example, increased vascular permeability, induced bronchial smooth muscle contraction or mucus production, and neutrophil chemotaxis. Therefore, it is necessary to reduce the release of mediators to prevent and / or alleviate allergic symptoms. As shown in Figures 1A and 1B, the cytotoxic effect of the phlorotannin compound on the test cell line was not significant. The invention may not have been affected by toxic effects by the test compound. In the present invention, the inhibitory effects of phlorotannin compounds isolated from Ecklonia cava were evaluated using human basophil leukemia (KU812) and rat basophil leukemia (RBL-2H3) cell lines.

As a first consideration, the active compounds Compounds 2 and 3 showed the most potent and dose dependent inhibitory effect on histamine released from each cell line and each stimulant. In antibody facilitating treatment, Dieckol, compound 2, exhibited the highest inhibitory activity (IC50 = 27.8 μM) against KU812 cells, whereas 6,6′-bieckol, compound 3, was associated with the degranulation level of RBL-2H3 cells. Most effective inhibitory effect (IC 50 = 38.76 μM) (see Table 2).

TABLE 2

Inhibitory Effect of Phlorotannin on Degranulation

a: no activity

In each case, the antibody-promoted active compound showed a stronger degranulation inhibitory effect compared to the treatment with A23187 as shown in FIGS. 3A and 3C. This evidence suggests that active compounds may be useful in preventing allergen-IgE-induced allergic reactions. These results also suggest a multifunctional inhibitory effect of the phlorotannin compound on hydramine release. In all these cases, phloroglucinol having the smallest molecular weight, compound 1, showed no inhibitory effect on histamine release in each cell line. Compound 4 with an intermediate molecular weight showed a moderate inhibitory effect comparable to the other two active compounds. Compounds 2 and 3 with higher molecular weights showed stronger activity, indicating that the molecular size or number of phenol groups can be an important factor in exerting their activity. The same evidence has also been reported for apple polyphenols.

Calcium ion ^permeators selectively increased intracellular Ca ²⁺ to mediate histamine release, which also caused degranulation in mast cells and basophils. This increase may correspond to an increase in cell membrane permeability. In the present invention, isolated fluorologusinol compounds were able to inhibit histamine release promoted by A23187 in each cell line. A possible explanation for this phenomenon is that phlorotannin mitigates the increase in intracellular Ca ²⁺ levels corresponding to a decrease in histamine release from cells by stabilizing the cell membrane.

Interactions between green tea derived catechins and polyphenolic compounds have also been reported. It has been reported that green tea-derived catechins can inhibit membrane leakage and make the membrane structure dense.

In addition, the degranulation inhibitory effect of phlorotannin compounds by antibody promotion suggested other possible mechanisms. The mechanism of inhibition of green tea catechins has been studied closely. Epigallocatechin galleate (EGCg) inhibited tyrosine phosphorylation of protein kinases involved in RBL-2H3 degranulation. EGCg also inhibited the expression of FcεRI in KU812F cells. It has also been reported that phlorotannin compounds have an inhibitory effect on hyalurodinase enzymes. The mechanism presented may be due to the inhibitory effect of the phlorotannin compound on histamine release.

Moreover, flow cytometry provided strong evidence that test compounds (2, 3 and 4) were able to inhibit binding between IgE and FcεRI receptors in a dose dependent manner (see FIG. 4). Inhibition of binding between IgE and FcεRI receptors is a target for the development of anti-allergic drugs because the crosslinking of FcεRI induced by complex formation of IgE and allergens is a determining step in the IgE-mediated allergic response pathway.

Previous reports suggest that monoclonal antibodies specific for IgE are effective adjuvant therapies for asthma. There is also evidence that the binding of monomeric IgE to FcεRI in mast cells and basophils not only promotes survival and proliferation of these cells but also increases the expression of FcεRI receptors on the cell surface even when they do not induce cell stimulation. have. On the other hand, compounds 2 and 4 exhibited a strong inhibitory effect on the binding activity between IgE and its receptor at the highest concentration, whereas compound 3, 6,6'-bieckol, having the same structure and molecular weight, showed a lower inhibitory effect. This indicated that various structures with less hydroxyl functionality play an important role in inhibiting the binding between IgE and its receptor. On the other hand, compound 3 also has the same inhibitory effect on IgE promoted degranulation (see FIG. 3) at the highest concentration (100 μg / mL) compared to compound 2, which presents another option for the anti-allergic effect of these compounds. Indicated.

In a previous study, green tea polyphenols, (-)-epigallocatechin-3-O-gallate, inhibited mast cell RBL-2H3 degranulation through binding to lipid raft 67RL on the cell surface. It was. Other studies have revealed that inhibition of histamine release from RBL-2H3 cells by EGCg was mediated by inhibiting tyrosine phosphorylation of antigens and proteins induced by A23187. Inhibition of elevated intracellular Ca ²⁺ via FcεRI activation is also a possible mechanism.

As a result, the present invention revealed that phlorotannins isolated from brown algae, ecchi, exhibited anti-allergic activity. Among the four single compounds, dieckol and 6,6'-bieckol showed the strongest inhibitory effect. One possible mechanism of degranulation inhibition is that the test compound inhibits the binding between the IgE and the FcεRI receptor. This suggests that compounds 2, 3 and 4 may be potential candidates in the pharmaceutical and food industries. Furthermore, the study of other mechanisms supporting the anti-allergic effects of these compounds should provide clearer evidence for the end purpose described above.

Claims (5)

1. A food composition having an anti-allergic activity containing an ectopically derived phlorotannin compound as an active ingredient.
2. Cosmetic composition having an anti-allergic activity containing as an active ingredient a chlorotannin derived from Ecklonia cava.
3. A pharmaceutical composition having an anti-allergic activity containing an ectopically derived phlorotannin compound as an active ingredient.
4. The method of claim 1, wherein the phlorotannin compound is phloroglucinol, dieckol, 6,6'-bieckol or 1- (3 ', 5'-dihydro-xyphenoxy) -7- (2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1,4-dioxin [1- (3 ', 5'-dihydro-xyphenoxy) -7- ( 2 ", 4", 6-trihydroxyphenoxy) -2,4,9-trihydroxydibenzo-1,4-dioxin].
5. The method of claim 3, wherein the allergic disease to which the anti-allergic active pharmaceutical composition can be applied includes, as a respiratory disease, bronchial asthma, hyperthermia, vasomotor nerve rhinitis, hypertrophic rhinitis, allergic bronchitis, Loepller syndrome ( Transient lung infiltration), dietary allergic gastritis as a digestive system disease, allergic diarrhea, allergic stomatitis, entero purpura, circulatory disease as periarterial arteritis, obstructive endocarditis, angina pectoris, endocarditis, skin Hives, quinque edema (vascular neuroedema), nodular erythema, purpura, purpura, sympathetic ophthalmitis, allergic conjunctivitis, allergic keratitis, or diffuse glomerulonephritis or migraines Pharmaceutical composition, characterized in that.

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