JP5171066B2 - Anti-allergic action of propolis - Google Patents

Description

  The present invention relates to a prophylactic or therapeutic agent for allergy comprising an antiallergic active ingredient derived from propolis. The active ingredient derived from propolis suppresses the production or release of leukotriene, and is effective for allergic diseases such as hay fever such as Japanese cedar pollinosis, bronchial asthma and allergic rhinitis.

  Propolis is a glue-like substance made by mixing sap of trees collected by bees, shoots and exudates of plants with beeswax, etc., and has antibacterial, antioxidant, anti-inflammatory, and antitumor effects. It is known (patent documents 1 to 4).

On the other hand, the number of patients with bronchial asthma, hay fever typified by cedar and ragweed, and allergic rhinitis has been increasing in recent years. Has been.
JP2002-308830 JP-A-9-143179 JP 2004-159563 A JP-A-8-228701

  An object of the present invention is to prevent or treat bronchial asthma, allergic rhinitis, and hay fever by suppressing the production or release of leukotrienes.

  The present inventor searched for an active ingredient by paying attention to the production inhibitory effect or release inhibitory effect of propolis on leukotriene. As a result, Artepilin C and (E) -3- (2,2-dimethyl-8-prenyl-2H- The inventors have found that 1-benzopyran-6-yl) -2-propenoic acid, durupanin, buccalin and kaempferide have a strong leukotriene production inhibitory action or release inhibitory action, thereby completing the present invention.

The present invention provides the following leukotriene production inhibitor or release inhibitor, and a preventive or therapeutic agent for bronchial asthma / hay fever / allergic rhinitis.
1. A leukotriene production or release inhibitor comprising propolis or an extract thereof as an active ingredient.
2. A prophylactic or therapeutic agent for bronchial asthma comprising propolis or an extract thereof as an active ingredient.
3. A preventive or therapeutic agent for hay fever, comprising propolis or an extract thereof as an active ingredient.
4). Item 4. The preventive or therapeutic agent according to item 3, wherein the hay fever is cedar pollinosis.
5. A prophylactic or therapeutic agent for allergic rhinitis comprising propolis or an extract thereof as an active ingredient.
6). The active ingredient is selected from the group consisting of artepilin C, (E) -3- (2,2-dimethyl-8-prenyl-2H-1-benzopyran-6-yl) -2-propenoic acid, dourpanine, buccalin and kaempferide. Item 6. The leukotriene production inhibitor or release inhibitor according to any one of Items 1 to 5, which is at least one, or a prophylactic or therapeutic agent for bronchial asthma / hay fever / allergic rhinitis.

  According to the present invention, production or release of leukotriene can be suppressed by a component derived from propolis. Since leukotrienes strongly contract bronchial smooth muscle, the present invention is useful for preventing or treating bronchial asthma.

  Leukotriene is also deeply involved in hay fever or allergic rhinitis, and is useful as a preventive or therapeutic agent for hay fever or allergic rhinitis.

  In the present invention, the active ingredients derived from propolis having leukotriene inhibitory action (production suppression, release suppression, receptor antagonistic action, etc.) include the following:

  Note that (E) -3- (2,2-dimethyl-8-preny-2H-1-benzopyran-6-yl) -2-propenoic acid is 2,2-dimethyl-8-prenylchromene-6-propene. Although referred to as an acid, it is hereinafter referred to as “criphorin”.

  All of the above five active ingredients are contained in propolis.

  In the present specification, propolis is not particularly limited, and may be derived from any production area / plant such as Brazil, China, European countries, Oceania, USA, and the like. Therefore, any propolis such as aleklin, super green, eucalyptus, ultra green, etc. can be used, and among them, the alekrine propolis made in Brazil is particularly preferable.

  As the “propolis or an extract thereof” of the present invention, propolis itself can be used, but one in which the above five active ingredients are more concentrated than the propolis itself can be preferably used. Note that “the active ingredient is concentrated” means that the ratio of at least one of the five compounds to the whole is high. Specifically, in the process of extraction or purification, the five effective ingredients are used. The removal of substances other than the component compounds is exemplified. The most preferred “propolis or extract thereof” is a substance (single substance or mixture) in which at least one of the above five active ingredients has been purified and isolated.

The solvent for obtaining the propolis extract is not particularly limited as long as at least one of the above five substances can be extracted. For example, water, CO ₂ , methanol, ethanol, n-propanol, isopropanol, n-butanol, etc. Esters such as lower alcohol and ethyl acetate, ketones such as acetone and methyl ethyl ketone, halogenated hydrocarbons such as methylene chloride and chloroform, aromatic hydrocarbons such as toluene and xylene, ethers such as tetrahydrofuran, diethyl ether and diisopropyl ether And glycols such as ethylene glycol and propylene glycol, glymes such as glyme and diglyme, cellosolves such as methyl cellosolve and ethyl cellosolve, DMF, DMSO and the like.

  The method for obtaining the propolis extract is not particularly limited. As the propolis as a raw material, a propolis bulk or a propolis bulk washed with alcohol is usually used. These raw propolis ingots may be used as they are, but it is preferable to use a propolis that has been cut or pulverized to an appropriate size because the extraction efficiency is improved.

  The extraction time is not particularly limited, and can be appropriately set depending on the form of propolis used as a raw material, the type and amount of a solvent used for extraction, the temperature at the time of extraction, stirring conditions, and the like. After completion of the extraction operation, the solid matter can be removed from the extraction mixture by a normal method, for example, filtration, and further, if necessary, a propolis solvent extraction fraction can be obtained by centrifugation.

  The propolis solvent extract fraction thus obtained can be used as it is as an active ingredient of the present invention, but the form of the concentrate obtained by concentrating the extract fraction by a conventional method or the spray of the concentrate is used. It can also be used in the form of a powder obtained by drying and freeze-drying.

  The five compounds of the active ingredient of the present invention may be in a free acid form or a salt form. Examples of the salt include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.), and preferably alkali metal salts. Such a salt can be easily converted into a salt form by reacting the compound with an alkali metal hydroxide, an alkaline earth metal hydroxide or the like.

  The active ingredient of the present invention can further purify propolis or an extract thereof by solvent extraction, column chromatography, recrystallization, liquid chromatography or the like.

The active ingredient of the present invention suppresses the action of leukotriene by mechanisms such as leukotriene production inhibition, release inhibition, leukotriene binding inhibition, and leukotriene binding. Examples of the leukotriene (LT) to be suppressed include LTC ₄ , LTD ₄ , LTE _{4 and the} like.

  For example, in the case of propolis or propolis extract, the effective daily dose for adults is about 10 mg to 2 g, preferably about 100 mg to 1 g, more preferably about 150 to 500 mg, and most preferably about 300 mg. These can be taken once a day or divided into 2 to 4 times. When five active ingredients of propolis are isolated, the effective amount of each active ingredient is about 1 μg to 100 mg.

  Although the active ingredient of the present invention is desirably administered to humans, it can be similarly administered to animals / pets such as dogs and cats and domestic animals such as horses, cattle, pigs and poultry, as well as bronchial asthma, hay fever and allergies. Rhinitis can be prevented or treated. The active ingredient of the present invention can be taken as an oral ingestion preparation. The active ingredient of the present invention is usually used in the form of a liquid preparation or solid preparation suitable for oral administration.

  Examples of liquid preparations include liquids, drinks, and syrups, and solid preparations include powders, granules, capsules, tablets, chewables, lozenges and the like for oral administration.

  In producing a liquid preparation, water, glycerin, propylene glycol, simple syrup, ethanol, ethylene glycol, polyethylene glycol, sorbitol and the like can be used as excipients.

  When producing solid preparations, excipients such as lactose, sucrose, glucose, corn starch, gelatin, starch, dextrin, calcium phosphate, calcium carbonate, synthetic or natural aluminum silicate, magnesium oxide, dried Aluminum hydroxide, magnesium stearate, sodium bicarbonate, dry yeast and the like can be used.

  Furthermore, the active ingredient of the present invention includes, as desired, stabilizers such as citric acid, phosphoric acid, malic acid or salts thereof; high-intensity sweeteners such as sucralose and acesulfame potassium; sucrose, fructose and honey Sweeteners; preservatives such as alcohols, glycerin; usual additives such as diluents, buffers, flavoring agents and coloring agents may be added.

  The active ingredient of the present invention is a food intended for health, health maintenance, enhancement, etc., for example, health food, functional food, dietary supplement, supplement, or food for special use defined by the Ministry of Health, Labor and Welfare (for example, food for specified health use, nutrition Functional food, food for the sick, combination food for the sick, food for the elderly, etc.).

Hereinafter, the present invention will be described in more detail based on examples.
Production Example 1
Artepilin C and cryforin were isolated according to the following method.
(i) The ethanol extract of Aleklin propolis bulk (Minas Gerais State) was concentrated with a rotary evaporator, and when the viscosity increased, it was switched to a vacuum drying method and dried at room temperature overnight.
(ii) Propolis extract (3.3 g) after drying was subjected to column chromatography (gel type: silica gel Wako gel C-200, column size: φ5 × 40 cm, elution solvent: ethyl acetate), and n-hexane. Elution was performed sequentially with ethyl acetate, chloroform, ethanol, and methanol. When the leukotriene release inhibitory action was examined on each of the obtained components, the main active substance was found in the ethyl acetate eluate.
(iii) The above extract was subjected to column chromatography (gel type: silica gel Wako gel C-200, column size: φ5 × 40 cm, elution solvent: 1%, 2%, 3%, 4%, 5%, 6%, Purify with n-hexane containing 8% ethyl acetate, elution conditions: 200 ml each for 20 ml fraction), and 9 fractions (Fr 4-6; 28 mg, Fr 7-12; 53) mg, Fr 15-17; 23 mg, Fr 18-24; 105 mg, Fr 25-32; 105 mg, Fr 33-41; 340 mg, Fr 42-49; 393 mg, Fr 50-79; 622 mg, Recovery; 310 mg). As a result of examining the leukotriene release inhibitory action on the obtained 9 fractions, the main active body was found in Fr. 25 to Fr.
(iv) Fr. 25 to 32, which had the strongest activity, were subjected to liquid chromatography (model: 880-PU system manufactured by JASCO, column: Cosmosil 5C18-AR-II 10 × 250 mm, manufactured by Nacalai Tesque, moving bed: solution A: 0.05% trifluoroacetic acid, B solution: acetonitrile, A: B = 50:50 (v / v) to 10:90 (v / v) 15 minutes linear gradient, 10:90 (v / v) held for 5 minutes And detection wavelength: 254 nm, flow rate: 2.5 ml / min) to obtain Artepiline C (208 mg) and Clifolin (10.4 mg).

The identification results of Artepilin C and Clifolin are shown below.
Measuring device NM R; AVNCE 50 0 type MS manufactured by Bruker Biospin; JMS JT-T 100 LC type manufactured by JEOL

(i) Artepilin C
EI-MS m / z: 3 0 0 “M” ⁺ , CI − MS m / z: 3 0 1 [M + H] ^{+
1 H - N M R (5} 0 0 M H z; C D 3 O D): δ 1. 7 3 (6 H, s, a - C H 3 × 2), 1. 7 6 (6 H, s, a - C H 3 × 2), 3. 3 1 (4 H, m, g - C H 2 × 2: overlaps with measurement solvent), 5. 3 2 (2 H, m, H c × 2), 6. 2 (1 H, d, J df = 16 Hz, H d), 7. 14 (2H, s, He), 7. 5 3 (1 H, d, J df = 16 H z, H f)

(ii) Clifolin ESI-MS (negative mode) m / z: 297 [M−H] ^−
1 H-NMR (500 MHz; CD ₃ OD): δ 1.42 (6H, s, a-CH ₃ × 2), 1.72 (3H, s, b-CH ₃ ), 1.73 (3H, s, _{b-CH 3), 3.25 (} 2H, d, Jcd = 8Hz, c-CH 2), 5.25 (1H, m, d-CH), 5.72 (1H, d, Jeg = 10Hz, He ), 6.25 (1H, d, Jfi = 16 Hz, Hf), 6.38 (1H, d, Jeg = 10 Hz, Hg), 7.11 (1H, d, Jhh ′ = 2 Hz, Hh), 7. 19 (1H, d, Jhh ′ = 2 Hz, Hh ′), 7.55 (1H, d, Jfi = 16 Hz, Hi)

Production Example 2
In accordance with the following method, durpanine, buccalin and kaempferide were isolated and identified.
(i) A 80% ethanol extract of 2 g of Alecrin propolis bulk (Minas Gerais State) was concentrated and dried with a rotary evaporator.
(ii) The dried propolis extract (1.3 g) was partitioned between ethyl acetate and water to obtain an ethyl acetate section (970 mg).
(iii) Column chromatography of ethyl acetate (726 mg) (gel type: silica gel Yamazen Hi Flash ^™ , column size: φ26 × 100 mm, elution solvent: 2%, 5%, 10%, 20%, 40% Purify with chloroform containing ethyl acetate, elution conditions: 200 ml each and 20 ml each), and eleven fractions (Fr 4-5; 29.4 mg, Fr 6-7; 20.2 mg, Fr 8- 10; 139.2 mg, Fr 11-13; 133.0 mg, Fr 14-17; 57.6 mg, Fr 18-20; 9.6 mg, Fr 21-25; 25.5 mg, Fr 26-31; 34.3 mg, Fr 32-40; 39.0 mg, Fr 41-45; 18.9 mg, Fr 46-50; 62.6 mg).
(iv) Fraction 26-31 was fractionated by thin layer chromatography (gel type: silica gel 60 F254, developing solvent: ethyl acetate: chloroform = 1: 1, detection: 254 nm), and showed a Rf value of 0.43 Got. Furthermore, liquid chromatography (model: Waters 600 series, column: Cosmosil 5C18-AR-II 10 × 250 mm, manufactured by Nacalai Tesque, moving bed: solution A: 0.1% trifluoroacetic acid (5 min), solution B: acetonitrile, A: B: 50:50 (v / v) for 5 minutes, then A: B = 50: 50 (v / v) to 10:90 (v / v) 30 minute linear gradient), detection wavelength: 260 nm, flow rate : 1 ml / min) to obtain durpanine (3.1 mg).
(v) Fraction 11-13 in medium pressure column (gel type: ODS-SM 26 × 100 mm, elution solvent: 0.1% trifluoroacetic acid containing 70%, 80%, 90%, 100% methanol, elution conditions: 7 fractions (Fr 1-9; 12.9 mg, Fr 10-13; 50.3 mg, Fr 14-20; 8.4 mg, Fr 21-25; 34 mg, Fr 26-30; 23.1 mg, Fr 31-45; 8.0 mg, Fr 46-60; 7.1 mg).
(vi) After adding a few drops of acetone to fraction 26-30, hexane was added to the extent that the solution was sloppy and allowed to stand at room temperature to obtain colorless needle-like crystal buccalin (9.8 mg).
(vii) Methanol was added to fractions 10-13 and allowed to stand to obtain kaempferide (24.4 mg) as yellow needle crystals.

The identification results of durpanine, baccarain and kaempferide are shown below.
Measuring device mass spectrometer: JMS-AX505 manufactured by JEOL
Nuclear porcelain resonance system: JCP ECP-500 NMR

Durpanin
EIMS (m / z): m / z 232 [M] ⁺ (81.3%), 177 (100)
¹ H NMR (Acetone-d ₆ ): d _H 1.72 (6H, s), 3.32 (2H, d, 7.4 Hz), 5.35 (1H, m), 6.28 (1H, d, 15.8 Hz), 6.89 (1H, d, 8.3 Hz), 7.32 (1H, dd, 2.1, 8.3 Hz), 7.39 (1H, d, 2.1 Hz), 7.55 (1H, d, 15.8 Hz)

Baccaroin
EIMS (m / z): m / z 364 [M] ⁺ (8.2%), 232 (100), 214 (25.8), 177 (34.8), 105 (31.6), 91 (38.4)
¹ H NMR (CDCl ₃ ): d _H 1.67 (3H, s), 1.76 (3H, s), 2.92 (2H, dd, 7.6, 8.0 Hz), 3.09 (2H, dd, 7.6, 7.8 Hz), 3.15 ( 2H, d, 7.1 Hz), 5.18 (1H, m), 6.38 (1H, d, 16.1 Hz), 6.98 (1H, d, 8.7 Hz), 7.28-7.40 (7H, m), 7.37 (1H, d, 16.1 Hz)

Kaempferide
EIMS (m / z): 300 [M] ⁺ (100%), 299 (31.3), 285 (34.8), 232 (43.4)
¹ H NMR (Acetone-d ₆ ): d _H 3.89 (3H, s), 6.36 (1H, d, 2.1 Hz), 6.53 (1H, d, 2.1 Hz), 7.11 (1H, ddd, 2.1, 3.0, 9.2 Hz), 8.22 (1H, ddd, 2.1, 3.0, 9.2 Hz)

Example 1: LT Release Inhibition According to the following procedure, the LT release inhibitory action of propolis-derived artepilin C and the like was examined.

Kit used: Leukotriene concentration measurement ELISA Kit (kit name CAST (registered trademark) -2000: The BUHLMANN)
Other: Solution for cleaning: (HEPES-Tyrod's BSASolution, Ca2 + excluded, pH = 7.4)
Solution for test substance preparation: (HEPES-Tyrod's BSASolution, with antibiotics, pH = 7.4)
Calcium Ionophore A23187 (Sigma): final concentration 10μmol / L
Preparation of test substance: Prepare each test substance to a predetermined final concentration.
Calcium Ionophore A23187 is dissolved in 1 ml DMSO and diluted 317 times with Ca-free HEPES-Tyrode's.
Cell extraction procedure: A 9-week-old rat is exsanguinated by decapitation. Immediately, 24 mL of physiological saline is injected into the abdominal cavity, the abdomen is lightly massaged, and the infusion is collected after laparotomy. Then wash three times with Stimulation Buffer. After washing, float the cells in 1 mL of Stimilation Buffer, count the number of cells, and centrifuge. Remove supernatant and adjust to 2 × 10 ⁴ cells / mL with Stimulation Buffer. After preparation, aliquot 200 μL cell suspension into a 1.5 mL tube, and measure and release LT using the leukotriene concentration measurement ELISA Kit as follows.
LT release: Aliquot 200 μL of cell suspension.
Back ground (BG)
200μL: Cell suspension
50μL: Solition for test substance preparation
50μL: Stimulation Buffer
For Positive Control (PC)
200μL: Cell suspension
50μL: Solition for test substance preparation
50μL: Final concentration 10μmol / L Calcium ionophore solution
For Sample
200μL: Cell suspension
50 μL: Each sample solution (prepared to final concentration)
50μL: Final concentration 10μmol / L Calcium ionophore solution
Stir gently after mixing each. After stirring, incubate at 37 ° C for 40 minutes. After incubation, cool with ice, stop the reaction, and centrifuge (1000 x G, 4 ° C, 3 minutes). Collect the supernatant after centrifugation and measure the LT concentration using the collected supernatant.
Dilute 32 times with ELISA Buffer when measuring absorbance

Leukotriene release inhibition rate (%)

The results are shown below.

Example 2
In accordance with the following procedure, the LT release inhibitory action of propolis-derived durpanin, buccalin, and kaempferide was examined. As a positive control, phorbol myristate acetate (PMA) was used.

Diluent: 137 mM NaCl, 2.68 mM KCL, 0.5 mM MgCl ₂ , 0.9 mM CaCl2, 8.1 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ (pH 7.4)
Calcium Ionophore A23187 (Sigma): final concentration 5μmol / L
Cell suspension: Differentiated HL-60 cells 6 × 10 ⁵ cells / well
Assay volume: 250 μl
Incubate the cell suspension at 37 ° C for 15 minutes, add each sample solution, PMA solution or water, and incubate at 37 ° C for 15 minutes. After incubation, ice-cool at 4 ° C for 5 minutes to stop the reaction, and then centrifuge (250 x G, 4 ° C, 5 minutes). Collect the supernatant after centrifugation and measure the LT concentration using the collected supernatant.

The results are shown in Table 2 below.

Formulation Example 1
200 ml of the propolis ethanol extract was sprayed and dried into 1 kg of lactose, powdered, and filled into hard capsules to obtain propolis ethanol extract-containing capsules.

Formulation example 2
50ml ethanol extract of propolis enriched with artepilin C
Minamata 250g
300g sugar
50ml water
250 g of chickenpox, 300 g of sugar, and 50 ml of water are dissolved by heating, and 50 ml of ethanol extract of propolis enriched with artepilin C is added. In particular, it was molded into a suitable size to obtain a propolis ethanol extract-containing candy.

Formulation Example 3
Propolis hydrous ethanol extract 0.5ml
Ascorbic acid 300mg
Honey 10g
Lemon juice 10ml
A proper amount of water The above ingredients were mixed to obtain a total amount of 50 ml of a propolis-containing drink containing an ethanol extract.

Formulation Example 4
A supplement was prepared according to the following formulation using an 80% ethanol extract of propolis enriched with cryophorin.
The powder of the following prescription was uniformly mixed, and a 160 mg tablet supplement was prepared by a conventional method using a tableting machine.
Spray dried product of ethanol extract of propolis enriched with cryophorin 10 mg
Dry beer yeast powder 50 mg
Reduced maltose 75 mg
Sucrose fatty acid ester 20 mg
Cellulose 5 mg

Formulation Example 5
Tablets were prepared according to the following formulation using a freeze-dried 10% ethanol extract of propolis enriched with dulpanine.
Spray dried product of ethanol extract of propolis enriched with dulpanine 10 mg
Microcrystalline cellulose 80 mg
Corn starch 78 mg
Magnesium stearate 2 mg

Formulation Example 6
As a coating agent, 32.15 g of corn starch was added (coating) to 75 g of ethanol extract enriched with baccharin to obtain 107.15 g of coated granules of ethanol extract. In addition, the coating ratio of this granule is 0.3.

Formulation Example 7
According to a conventional method, 50 μg of an ethanol extract enriched with kaempferide was mixed with 150 mg of soybean oil, 20 mg of beeswax and 30 mg of glycerin fatty acid ester and filled into a gelatin soft capsule to obtain a granular health food (this oral composition).

Claims (5)

Leukotriene containing (E) -3- (2,2-dimethyl-8-prenyl-2H-1-benzopyran-6-yl) -2-propenoic acid (cryphorin) or a salt thereof as an active ingredient Production or release inhibitor. Bronchial, containing isolated or purified (E) -3- (2,2-dimethyl-8-prenyl-2H-1-benzopyran-6-yl) -2-propenoic acid (cryphorin) or a salt thereof as an active ingredient Preventive or therapeutic agent for asthma. Pollen containing isolated or purified (E) -3- (2,2-dimethyl-8-prenyl-2H-1-benzopyran-6-yl) -2-propenoic acid (cryphorin) or a salt thereof as an active ingredient Preventive or therapeutic agent. The preventive or therapeutic agent according to claim 3, wherein the hay fever is cedar pollinosis. Allergy with isolated or purified (E) -3- (2,2-dimethyl-8-prenyl-2H-1-benzopyran-6-yl) -2-propenoic acid (criphorin) or a salt thereof as an active ingredient Preventive or therapeutic agent for rhinitis.