JPH0920672A - Antiallergic substance, its production, antiallergic agent and functional food - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antiallergic substance by purifying matter found in Perilla frutescens crispa and having antiallergic action and specifying the active ingredient therein, to provide a production method for this antiallergic substance, and to obtain an antiallergic agent and functional food each containing this antiallergic substance. SOLUTION: Perilla frutescens crispa leaves are subjected to hot water extraction followed by solid-liquid separation, a precipitant is added to the resultant aqueous solution, water-soluble components are then leached from the resultant precipitate and then subjected to at least one kind of column chromatography to accomplish fractionation and purification, thus obtaining the objective substance having mast cell degranulation-inhibitory activity. This substance contains polysaccharides including amino acids and having a molecular weight of 13500 (antiallergic substance I), polyphenols leachable with 50% acetone by a column chromatography using a DEAE anion column (antiallergic substance II), and polyphenols leachable with 50% acetone containing 0.1N Hcl (antiallergic substance III).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antiallergic substance having mast cell degranulation inhibitory activity obtained from a perilla hot water extract, a method for producing an antiallergic substance from a perilla hot water extract, and an antiallergic substance. Antiallergic agents, including
And with respect to functional food, the anti-allergic substance of the present invention,
It is useful for treating and preventing allergies.

[0002]

2. Description of the Related Art Allergic diseases are classified into four types, type I to type IV, according to the type of antigen-antibody reaction. However, the usual allergic diseases nowadays are allergic bronchial asthma, allergic rhinitis, which is type I allergy.
Often refers to hay fever, atopic dermatitis, etc.

The mechanism of allergy is divided into three stages. In the first step, antigens such as pollen and house dust adhere to the mucous membrane of organs and produce IgE antibodies.
In the second step, chemical mediators such as histamine are released by degranulation from antigen-sensitized mast cells. In the third step, the chemical substances such as histamine released in the second step act on the organs to cause allergic symptoms.

In the treatment of the above-mentioned allergic diseases, antihistamines, steroids, degranulation inhibitors and the like are used. As an outline of the mechanism of action of these therapeutic agents,
Antihistamines can inhibit the action of histamine which is the third step, steroids can mainly inhibit the antibody production which is the first step, and degranulation inhibitors can inhibit the second step, degranulation from mast cells. Are known.

By the way, the hyaluronidase inhibitory activity is
Kakegawa et al., “J. Pharm. Dyn., 7, S-,” is one of the mechanisms that has an inhibitory effect on histamine release from mast cells.
96 (1984) ”. In addition, there is a correlation between hyaluronidase inhibitory activity and inhibition of degranulation from mast cells by Kakegawa et al. “Chem.Pharm.Bull.33 (11) 50”.
79-5082 (1985) ”, and a test using tannin was made clear.

Since the conventional antiallergic agents are often used regularly and are often administered to infants, safe therapeutic agents with few side effects have been conventionally demanded. Therefore, until now, various plants have been searched for antiallergic components, and it has already been confirmed that components contained in Lamiaceae plants have antiallergic effects.

For example, Japanese Patent Laid-Open No. 6-293652 discloses that perilla leaves extracted with an organic solvent have type I allergy suppressing effect. Japanese Unexamined Patent Publication No. 1-102027 discloses that callus derived from perilla leaves is induced, and the callus obtained by tissue culture is dried at 10 ° C.
It is disclosed that the extract is concentrated to dryness by extraction with 50% ethanol water at 50 ° C., and that this extract has mast cell degranulation inhibitory activity. JP-A-6-62795 discloses a perilla leaf extract obtained by extracting perilla leaves with a solvent such as water and alcohol and evaporating the solvent to dryness, and α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. Disclosed is an anti-allergic food containing a fatty acid containing a fatty acid as a constituent fatty acid. Japanese Patent Laid-Open No. 4-798
JP-A-52-52 discloses that the leaves and leaves of a Lamiaceae plant are ground, extracted with an organic solvent, and then the solvent is removed to obtain an allergy-improving food.

[0008]

However, the perilla extract having anti-allergic activity shown in each of the above prior arts is a perilla plant extracted with a solvent, and the solvent is evaporated to dryness. It has not been specified what kind of ingredients in them have an anti-allergic effect. Therefore, the present invention is to purify a substance having an antiallergic action contained in a perilla plant, to specify its components, to provide the specified antiallergic substance and a method for producing the same, and to provide the antiallergic substance. Anti-allergic agents, including
Another object of the present invention is to provide a functional food containing the antiallergic substance.

[0009]

[Means for Solving the Problems] As described above, it is known that the mast cell degranulation inhibitory activity and the hyaluronidase inhibitory activity are related to each other. Therefore, it is necessary to measure the hyaluronidase inhibitory activity. It is useful for searching active substances. Therefore, the present inventors have completed the present invention by conducting a search using the hyaluronidase inhibitory activity as an index and determining a component showing an inhibitory activity against degranulation from mast cells from a hot water extract of perilla leaf.

That is, the first anti-allergic substance of the present invention is obtained from a perilla hot water extract, has a mast cell degranulation inhibitory activity, and has a molecular weight of 13,500 containing sugars and amino acids as constituent elements ( Antiallergic substance I)
It is characterized by being.

An anti-allergic substance other than the above-mentioned anti-allergic substance I of the present invention is characterized by being a polyphenol obtained from a perilla hot water extract and having mast cell degranulation inhibitory activity. The antiallergic substance which is the polyphenol of the present invention can be further classified into two types,
One can be eluted with 50% acetone by column chromatography using a DEAE anion column (the substance eluted by this method is used as an anti-allergic substance II
On the other hand, it can be eluted with 50% acetone containing 0.1N HCl by column chromatography using a DEAE anion column (the substance eluted by this method is referred to as antiallergic substance III).

The method for producing an anti-allergic substance of the present invention comprises:
Perilla leaves were extracted with hot water, solid-liquid was separated, the obtained aqueous solution was precipitated with a precipitant, the water-soluble component was eluted from the obtained precipitate, and one or two of the obtained eluted components were further extracted. It is characterized in that a substance having mast cell degranulation inhibitory activity is obtained by separating and purifying by column chromatography of at least one species.

The anti-allergic agent of the present invention is characterized in that an anti-allergic substance separated in each purification step is formulated.

The functional food of the present invention is characterized in that an anti-allergic substance separated in each of the above-mentioned purification steps is added to the food to impart an anti-allergic function to the food.

The present invention will be described in more detail below.

Perilla, which is a raw material for producing the antiallergic substance of the present invention, is a plant of the Labiatae family and its related plants. , Mint, swordfish, sesame, catarrhalius, columbine, sage, sage, thyme, basil, mint, lavender, oregano, seboli, lemon palm, rosemary, catnip, hyssop, bergamot. Leaves of these ingredients,
Stems and seeds are used and can be used in both raw plants and dried plants, but the dried ones are convenient raw materials in that they are highly storable.

In the method of obtaining an active ingredient from these perilla, it is essential to first extract perilla leaves with hot water in order to improve the extraction efficiency. The method of hot water extraction in the present invention can be carried out under appropriate conditions, but generally, for example, water is added to dried perilla leaves,
It is performed by boiling for about 15 minutes.

Further separation and purification of the active ingredient from the hot water extract obtained by the above boiling treatment can be carried out in accordance with the usual means for separating and purifying from natural products. For example,
After completion of hot water extraction, the solid and liquid are separated by filtration, centrifugation, etc., and the resulting aqueous solution is precipitated by using a precipitating agent such as cetylpyridinium chloride, and the water-soluble component is eluted from the precipitate, and the column is eluted. Chromatography [eg, column chromatography using DEAE ion exchange resin (manufactured by Tosoh Corporation)] can be used. By this column chromatography, the antiallergic substances I, II and III can be obtained by the elution method described above.

In order to further purify the antiallergic substances I, II and III of the present invention, it is necessary to prepare a carrier on which hyaluronidase is immobilized and to efficiently purify it by affinity chromatography using this carrier. The inventor found it.

The antiallergic substances I, II and III obtained according to the present invention have a mast cell degranulation inhibitory action and are used as pharmaceuticals or functional foods added to foods. In order to use the antiallergic substances I, II, and III of the present invention in pharmaceuticals or functional foods, it is possible to use any purified stage of the antiallergic substances I, II, and III. Is. For example, in functional foods, one or more of various purification stages of antiallergic substances I, II, and III are used in various foods, such as black tea, soft drinks, juices, candy, starch foods, and various processed foods. And the like, a functional food having a mast cell degranulation inhibitory action can be obtained. The anti-allergic substance according to the present invention can be used as an anti-allergic agent for the purpose of preventing or treating type I allergies such as allergic bronchial asthma, allergic rhinitis, hay fever, and atopic dermatitis. The antiallergic agents of the present invention include the antiallergic substances I, II and III at various purification stages.
It may be formulated as an active ingredient itself, or may be formulated and used in the form of coexistence with other antiallergic agents compatible with these antiallergic substances or other drugs.

The method of administration may be either oral or parenteral administration. Specific formulations include injections (eg, intravenous injection, intramuscular injection, subcutaneous injection, drip etc.), suppositories, tablets, powders, granules, capsules, creams, poultices and the like. As a carrier used in these preparations, an organic or inorganic inert carrier suitable for oral or parenteral use is used.
Specific examples include lactose, starch, vegetable or animal fats and oils. The ratio of the antiallergic substance of the present invention to the carrier in the preparation is 0.1 to 100%.
Can be varied between. Further, in this composition, a binder, an excipient, a lubricant, which is generally used in the formulation,
Additives such as a disintegrant and a wetting agent may be included.

The liquid preparation for oral use may be in any form such as an aqueous solution for internal use, a suspension, an emulsion and a syrup, and the composition thereof may contain additives and preservatives. it can.

The dose of the antiallergic agent of the present invention varies depending on the age, administration route, and number of administrations, and can be varied over a wide range. In this case, it is administered as a composition of an effective amount of a mast cell degranulation inhibitor, a suitable diluent, and a pharmacologically usable carrier. The effective amount administered is 0.5
μg to 50 mg / kg body weight / day, which is administered once to several times a day.

Molecular weight : Antiallergic substances I, II and III
When the molecular weight of each of these was measured by the ultrafiltration membrane method (Centriprep manufactured by Amicon), each of them passed through a filter having a molecular weight of 100,000, but did not pass through a filter having a molecular weight of 10,000. Is
It is confirmed that each has a molecular weight of 10,000 or more and 100,000 or less. Furthermore, when the molecular weight of the anti-allergic substance I is measured by an electrophoresis method using polyacrylamide gel (SDS-PAGA), a single band is observed at 13,500. Therefore, the molecular weight of the anti-allergic substance I is 1
It is set to 3,500.

The determination of the molecular weight by SDS-PAGA is performed as follows under reducing conditions. 95 samples
Denature by heat treatment at 5 ° C for 5 minutes, and use 0.06M Tris-HCl buffer (p
H6.8), 1.71% SDS, 6% glycerol,
A buffer having a composition of 0.1 M dithiothreitol and 0.002% BPB (abbreviation of bromophenol blue) was used, and after applying current for 5 minutes at 5 mA, 8-10 mA was applied.
Run for 150 minutes. After the migration, the protein is stained with silver and the polysaccharide is stained with PAS, and the standard molecular weight marker of the protein is simultaneously migrated to determine the molecular weight.

Solubility : Antiallergic substance I is readily soluble in water and methanol.

Both antiallergic substances II and III are slightly soluble in water.

None of the antiallergic substances I, II and III are extracted with neutral ethyl acetate. None of the anti-allergic substances I, II and III are extracted with neutral n-butanol.

In various ion exchange chromatography
Adsorption characteristics : Antiallergic substances I, II and III all adsorb on the DEAE anion exchange column. None of the anti-allergic substances I, II and III are acidic substances because they are not adsorbed on the carboxymethyl cellulose cation exchange resin (hereinafter referred to as CM cation exchange resin). The anti-allergic substance I adsorbed on the DEAE anion exchange column is eluted with a 1 M NaCl solution, and the anti-allergic substance II adsorbed on the DEAE anion exchange column is eluted with a 50% acetone solution. D
The antiallergic substance III adsorbed on the EAE anion exchange column is eluted with a 50% acetone solution containing 0.1N HCl.

Thermostability : Antiallergic substances I, II and II
Each of I is stable after heat treatment at 100 ° C for 30 minutes,
This heat treatment does not inactivate the mast cell degranulation inhibitory activity.

Enzyme treatment stability : antiallergic substances I and II
Neither of (3) and (III) inactivate the mast cell degranulation inhibitory activity by trypsin treatment at 30 ° C for 15 minutes.

Color reaction : The anti-allergic substance I is positive in the color reaction by the phenol-sulfuric acid method, and is thus shown to contain sugar.

Since the antiallergic substance I is positive by the carbazole-sulfuric acid method, it is understood that the sugar contained in the antiallergic substance I is a uronic acid-containing sugar in view of the above-mentioned properties.

The antiallergic substances II and III are precipitated when treated with Polyclar AT, and it is understood that they are polyphenols in view of the above-mentioned properties.

Substance Color : Purified anti-allergic substance I (obtained in Example 5 below) is brown.

Specific rotation : For purified antiallergic substance I (obtained in Example 5 below), [α] _D = -0.
4 (0.25, H ₂ O) UV absorption spectrum : A chart of the UV absorption spectrum of the purified antiallergic substance I (obtained in Example 5 below) is shown in FIG.

Infrared absorption spectrum : A chart of the infrared absorption spectrum of the purified anti-allergic substance I (obtained in Example 5 below) is shown in FIG.

Amino acid composition : 6N anti-allergic substance I
The results of amino acid analysis after hydrolysis with -HCl at 110 ° C for 24 hours are shown in Table 1 below. In this analysis, tryptophan cannot be detected.

[0039]

[Table 1]

Sugar composition : Antiallergic substance I was added to 2N-HC
Table 2 below shows the results of sugar composition analysis after hydrolysis at 100 ° C. for 4 hours.

[0041]

[Table 2]

LD ₅₀ : Purified antiallergic substance I (obtained in Example 5 below), antiallergic substance II (obtained in Example 1 below), antiallergic substance III
The LD ₅₀ of each (obtained in Example 1 below) is 2 g / Kg or more.

[0043]

【Example】

[Example 1] Antiallergic substance I (DEAE fraction I) from perilla leaves,
Anti-allergic substance II (DEAE fraction II) and anti-allele
Production of Gee Substance III (DEAE Fraction III) To 100 g of dried perilla leaves, 1 liter of water was added and boiled for 15 minutes. Next, this hot water extract was cooled and then suction-filtered under reduced pressure to separate solid and liquid. The resulting aqueous solution 500
Cetylpyridinium chloride is added to ml, and the temperature is 30 ° C.
A precipitate formed upon standing for 12 hours. The precipitate obtained is washed with 100 ml of 15% ethanol, 1
After removing the reagent by centrifugation at 20,000 rpm for 20 minutes, 50 ml of water was added to the precipitate to elute the water-soluble active ingredient.

The obtained eluate was packed in a DEAE-Toyopearl column (manufactured by Tosoh Corporation) equilibrated with 50 mM borate buffer (pH 7.4), washed with the same buffer, and then 1M NaCl was used as an eluent. Solution, 0.1N HC
1 solution, 50% acetone solution, containing 0.1N HCl 5
Elution was performed sequentially with a 0% acetone solution. Each of the collected fractions (DEAE fraction I, DEAE fraction II, DEAE
Fraction III) was dried under reduced pressure to obtain crude antiallergic substance I, antiallergic substance II and antiallergic substance III.

[Example 2] Measurement of hyaluronidase inhibitory activity : Perilla hot water extract obtained in Example 1 above, DEAE fraction I, DEAE fraction I
The hyaluronidase inhibitory activity of each of the I and DEAE fractions III was measured by the method described below according to the method described in "Sanitary Chemistry, 36 (4), 314-319 (1990)" by Sawabe et al.

2.83 mg of bovine testicular-derived hyaluronidase (manufactured by Sigma) was added to a buffer solution (0.1 M acetic acid, pH 4.
0) 0.15 ml of a solution dissolved in 1 ml and a buffer solution (0.1
0.2 ml of 0.3 M NaCl solution dissolved in M acetic acid, pH 4.0) was mixed and kept at 37 ° C. for 20 minutes.
Add 0.1 ml of each test solution to the reaction mixture,
It was kept warm for 0 minutes. Furthermore, 1.83 mg of hyaluronic acid potassium salt derived from rooster comb was used as a buffer solution (0.
0.2 ml of a solution dissolved in 1 ml of 1M acetic acid, pH 4.0
Was added and the mixture was kept warm at 37 ° C. for 20 minutes. Then 0.4N
1.83 mg of NaOH was added to a buffer solution (0.1 M acetic acid, pH
4.0) Add 0.2 ml of the solution dissolved in 1 ml, and add 37
After keeping the temperature at 20 ° C. for 20 minutes, the absorbance at 585 nm was measured.

The perilla hot water extract obtained in Example 1 above,
DEAE fraction I, DEAE fraction II, DEAE fraction III,
The result of measuring the hyaluronidase inhibitory activity using
The hyaluronidase inhibition rate is plotted on the vertical axis and each fraction is plotted on the horizontal axis, and the results are shown in FIG. In FIG. 3, the arrow (1) indicates 1M Na.
Cl solution injection timing, arrow (2) indicates 0.1N HCl
Injection time of solution, arrow (3) is injection time of 50% acetone solution, arrow (4) is 50% containing 0.1N HCl
The injection timing of the acetone solution is shown.

[Example 3] Measurement of mast cell degranulation inhibitory activity The perilla hot water extract and DEA in Example 1 were used.
The mast cell degranulation inhibitory activities of DEAE fraction I, DEAE fraction II, and DEAE fraction III eluted from E-Toyopearl column (manufactured by Tosoh Corporation) were measured by the following methods.

First, the rat peritoneal mast cells were prepared by the method of Nakagome [Kazuya Nakagome et al., Journal of Antibiolics 43.
Vol. 5, No. 5, 462-469, 1990). That is, in the abdominal cavity of Wistar rats,
20 ml of Tyrode solution was injected, and ascites was taken out with a pipette. The collected ascites was centrifuged at 4 ° C. and 100 × g for 12 minutes to collect the precipitated cells. Tyrode solution 2m
1 bovine serum albumin (abbreviation: BSA) physiological saline 4 ml suspended in 1
After centrifugation at 100 xg for 12 minutes at 100 ° C, the precipitated mast cells were collected. Anti-DNP mouse monoclonal IgE
By reacting the antibody at 37 ° C. for 1 hour, IgE was allowed to bind to mast cells to give a sensitized state. After washing several times with Tyrode's solution, the mast cells were suspended in Tyrode's solution containing 0.2% BSA so as to be 1 × 10 ⁶ cells / ml to prepare an IgE-sensitized mast cell suspension. It was

In the IgE-sensitized mast cell suspension obtained in the above step, the DEAE fraction I eluted from the perilla hot water extract in Example 1 and the DEAE-Toyopearl column (manufactured by Tosoh Corporation) was used. , DEAE Fraction II, and DE
After adding AE fraction III as a test solution and incubating at 37 ° C. for 5 minutes, it was used as a degranulation inducer and antigen (DNP-B
SA) (200 ng / ml) + phosphatidylserine (10 μg / ml) PBS (−) solution was added, and 3 again.
Incubated at 7 ° C for 10 minutes. 1500xg, 5
The amount of histamine contained in the supernatant after centrifugation for 1 minute was quantified by HPLC by post-column labeling with orthophthalaldehyde (abbreviation: OPA). The histamine release inhibitory activity was expressed as the inhibition rate with respect to the amount of histamine released by the degranulation inducer, and was calculated by the following formula (1).

Histamine release inhibition rate (%) = [1- (Hs-Hb) / (Hi-Hb)] × 100 Formula (1) Hb: Histamine amount released when cells are incubated only with PBS Hi: Cells Amount of histamine released when cells were incubated with degranulation inducer in the absence of test solution Hs: Histamine amount released when cells were incubated with degranulation inducer in the presence of test solution

Regarding the results obtained, DEAE-Toyope
FIG. 4 is a graph in which the horizontal axis represents the fraction number sequentially discharged from the arl column (manufactured by Tosoh Corporation) and the vertical axis represents the mast cell degranulation inhibition rate of each fraction. In FIG. 4, Fr. 1 is DEAE fraction I, Fr.
2 is DEAE fraction II, Fr. 3 indicates DEAE fraction III, the arrow in (1) indicates the injection timing of 1M NaCl solution,
The arrow in (2) indicates the injection timing of the 0.1N HCl solution, (3)
The arrow indicates the injection time of the 50% acetone solution, and the arrow in (4) indicates the injection time of the 0.1% HCl-containing 50% acetone solution. According to FIG. 4, DEAE fraction I, DEAE fraction
It can be seen that the II and DEAE fractions III have a smaller amount of free histamine than that of the perilla hot water extract, that is, have a high mast cell degranulation inhibitory activity.

Hot water extract, DEA in Example 1 above
DEAE fraction I, DEAE fraction II and DEAE fraction eluted from E-Toyopearl column (manufactured by Tosoh Corporation)
Regarding III, the hyaluronidase inhibitory activity was measured based on Example 2 and the mast cell degranulation inhibitory activity was measured according to Example 3 and the results are shown in Table 3 below.

[0054]

[Table 3]

The rate of hyaluronidase inhibition per volume of the above fractions is shown in Table 4 below as an indication of the degree of substance purification.

[0056]

[Table 4]

According to Tables 3 and 4, the substances having mast cell degranulation inhibitory activity obtained from perilla of the present invention have been previously reported for their hyaluronidase inhibition rate and mast cell degranulation inhibition rate. , It can be seen that they are almost correlated. It is also understood that the purification method of the present invention enhances mast cell degranulation inhibitory activity.

Example 4 Comparison between Water Extraction and Hot Water Extraction Dried perilla leaves were stirred with water at 4 ° C. for 18 hours and suction-filtered under reduced pressure to separate solid and liquid. The resulting aqueous solution 500
Cetylpyridinium chloride is added to ml, and the temperature is 30 ° C.
A precipitate formed upon standing for 12 hours. The precipitate was washed with 100 ml of 15% ethanol, 10,
After removing the reagent by centrifugation at 000 rpm for 20 minutes, 50 ml of water was added to the precipitate to elute the water-soluble active ingredient to obtain a water extraction filtrate.

On the other hand, the dried perilla leaves were washed with 100 ° C. water for 3 times.
After boiling for 0 minutes, the hot water extract was cooled and then suction filtered under reduced pressure to separate solid and liquid. In the following steps, the water-soluble active ingredient was eluted in the same manner as in the case of water extraction to obtain a hot water extraction filtrate.

The hyaluronidase inhibitory activity of the water extract filtrate and the hot water extract filtrate obtained in the above-mentioned steps was determined according to Sawabe et al.
According to the method described in “0)”, the measurement was performed by the following method.

2.83 mg of bovine testis-derived hyaluronidase (manufactured by Sigma) was added to a buffer solution (0.1 M acetic acid, pH 4.
0) 0.15 ml of a solution dissolved in 1 ml and a buffer solution (0.
0.3M NaCl dissolved in 1M acetic acid, pH 4.0)
0.2 ml of the solution was mixed and kept at 37 ° C. for 20 minutes. The reaction solution was prepared in two containers, and only one of the water extraction filtrate and the hot water extraction filtrate was added to each reaction solution, and the mixture was kept at 37 ° C. for 20 minutes. Furthermore, potassium salt of hyaluronic acid derived from cockle of rooster (manufactured by Sigma) 1.83 m
0.2 ml of a solution prepared by dissolving 1 g of 1 g in a buffer solution (0.1 M acetic acid, pH 4.0) was added, and the mixture was kept at 37 ° C for 20 minutes.

Subsequently, 0.1 ml of 0.4N NaOH and 0.1 ml of 0.8N boric acid solution adjusted to pH 9.1
Was added and reacted in a boiling water bath for 3 minutes. P-D for this
BA solution (1 g of p-dimethylaminobenzaldehyde,
3 ml of a solution obtained by mixing 1.25 ml of 10N hydrochloric acid and 98.75 ml of acetic acid) was added and mixed, and the mixture was kept at 37 ° C. for 20 minutes and then the absorbance at 585 nm was measured.

As a result of calculating the inhibition rate of hyaluronidase from the respective absorbances obtained, the inhibition rate of hyaluronidase of the water extract filtrate was 42.3%, whereas the inhibition rate of hyaluronidase of the hot water extract filtrate was 73.7%. Met. By the way, since the hyaluronidase inhibitory activity and the mast cell degranulation inhibitory activity of the mast cell degranulation inhibitory ingredient contained in perilla are correlated, hot water extraction is more effective than water extraction as the mast cell degranulation inhibitory ingredient. It turns out that it is advantageous for extraction.

[Example 5] Purification of anti-allergic substance I (DEAE fraction I) Purification step 1: After concentrating the DEAE fraction I in Example 1 above with Centriprep (manufactured by Amicon), an equal amount of methanol was added. Was added to prepare an antiallergic substance I solution dissolved in a 50% methanol solution. When the obtained solution was applied to a silica gel column and then developed with a 50% methanol solution, an active substance having mast cell degranulation inhibitory activity was eluted.

Purification step 2: The active substance purified in the above-mentioned purification step 1 was concentrated under reduced pressure with an evaporator to give 0.01
% 3-[(3-Colamidopropyl) -dimethylammoniol] -1-propanesulfonate (abbreviation: CH
APS) aqueous solution as eluent, Cellulofine GC-
Gel filtration column chromatography using 700 (manufactured by Chisso Corporation) was performed. Elution with an aqueous CHAPS solution eluted the purified active material.

Purification step 3: The active substance obtained in the above-mentioned purification step 2 was further purified by affinity chromatography as follows in order to further enhance the mast cell degranulation inhibitory activity.

The carrier used for this affinity chromatography was produced as follows. AF-Tresyl Toyopearl [Toyopearl HW6 which is a filler for gel filtration based on hydrophilic vinyl polymer manufactured by Tosoh Corporation.
5 (trade name) active carrier in which tresyl chloride is introduced] 2 g, 0.1 M NaHCO ₃ -containing 0.5 M N
After suspending in 20 ml of aCl (pH 7.8) solution (referred to as A solution) and washing with the same solution, hyaluronidase 194
A solution prepared by dissolving mg in 5 ml of A solution was added, and the mixture was gently shaken at 4 ° C. for 24 hours. Then 0.5M N
Washing with 100 ml of aCl solution, followed by 0.1 M Tris-HCl buffer (containing 0.5 M NaCl) (pH 8.
0), followed by further washing with 0.1 M Tris-HCl buffer (containing 0.5 M NaCl) (pH 8.0), adding 40 ml of the same solution, and gently shaking at 4 ° C. for 4 hours. Thus, a hyaluronidase-immobilized-tresyltoyopearl carrier was produced.

The obtained hyaluronidase-immobilized-tresyl toyopearl carrier was added to 0.1 M NaCl containing 0.1 M
After buffering with an acetate buffer (pH 4.0), the immobilized carrier was subjected to the Cellulofine GC-700 of the purification step 2 described above.
(Manufactured by Chisso Corporation) was added with an active substance obtained by gel filtration column chromatography and then washed with the same buffer, and then 0.1 M containing 0.5 M NaCl was added.
The active substance was eluted with Tris-HCl buffer (pH 8.0). The obtained active substance was thoroughly dialyzed against water, and then concentrated and dried under reduced pressure with an evaporator to obtain a purified antiallergic substance I.

The effect of inhibiting mast cell degranulation on the concentration of the obtained purified anti-allergic substance I is shown in FIG. The inhibition rate on the vertical axis in FIG. 5 is defined in Example 4 above.

Example 6 Production of Tea Bag Commercially available black tea leaves were crushed, and the purified anti-allergic substance I obtained in Example 5 was added to the crushed product in an amount of 3% by weight. Mixed well. The mixture of these three kinds was packed in a tea bag to obtain a tea bag to which the purified antiallergic substance I was added. By exuding this tea bag with boiling water, the purified anti-allergic substance I was eluted and mixed with black tea, and black tea with mast cell degranulation inhibitory activity could be obtained.

[Example 7] Production of orange juice Three 1 kg of commercially available orange juice were prepared. To each orange juice, the purified anti-allergic substance I obtained in Example 5 and one of the anti-allergic substance II and anti-allergic substance III obtained in Example 1 were added so as to be 3% by weight. And mixed well to obtain three types of orange juice to which mast cell degranulation inhibitory activity was imparted.

[0072]

INDUSTRIAL APPLICABILITY The antiallergic substances (antiallergic substance I, antiallergic substance II, antiallergic substance III) obtained from the perilla hot water extract according to the present invention are excellent mast cell degranulation inhibitors described in the present specification. Shows activity. These substances have a high allowable intake amount and are useful as pharmaceuticals or functional foods to which the substances are added.

In the method for producing an anti-allergic substance according to the present invention, perilla leaves are extracted with hot water, and the active ingredient is fractionated and purified by one or more column chromatography. Therefore, mast cell degranulation is performed. The inhibitory activity can be increased.

Since the method for producing an anti-allergic substance according to the present invention is purified by affinity chromatography using a hyaluronidase-immobilized carrier in addition to the above-mentioned production method, mast cell degranulation inhibitory activity can be enhanced.

[Brief description of drawings]

FIG. 1 shows a chart of an ultraviolet absorption spectrum of a purified antiallergic substance I.

FIG. 2 shows an infrared absorption spectrum chart of purified anti-allergic substance I.

FIG. 3 Perilla hot water extract obtained in Example 1, DEAE
6 is a graph showing the results of measurement of hyaluronidase inhibitory activity using I, DEAE fraction II, and DEAE fraction III.

FIG. 4 Fraction number [DEA] sequentially discharged from a DEAE-Toyopearl column (manufactured by Tosoh Corporation)
E fraction I (anti-allergic substance I), DEAE fraction II (anti-allergic substance II), DEAE fraction III (anti-allergic substance III)] is plotted on the horizontal axis, and mast cell degranulation inhibition rate of each fraction is plotted on the vertical axis. The graph which shows the result.

FIG. 5 is a graph showing the mast cell degranulation inhibitory effect on the concentration of the purified anti-allergic substance I obtained in Example 5.

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[Procedure amendment]

[Submission date] October 11, 1995

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction contents]

The antiallergic substances I, II and III obtained according to the present invention have a mast cell degranulation inhibitory action and are used as pharmaceuticals or functional foods added to foods. Antiallergic substances I, II, II of the present invention
When I is used as a medicine or a functional food, it is possible to use any of the purified stages of antiallergic substances I, II, and III. For example,
In functional foods, antiallergic substances I, II, I
A mast cell degranulation inhibitory action is added by adding one or more of the various purification steps of II to various foods such as black tea, soft drinks, juices, candy, starchy foods, various processed foods, etc. It is possible to obtain functional foods. Antiallergic substances according to the invention, A barrel ghee bronchial asthma, A barrel ghee rhinitis, hay fever, as an antiallergic agent for the purpose of the Type I A barrel ghee such atopic dermatitis prevent or treat Can be used. The antiallergic agent of the present invention may be prepared by formulating the antiallergic substances I, II and III themselves at various purification stages as active ingredients, or other antiallergic agents or other pharmaceuticals compatible with these antiallergic substances. It can be formulated and used in the form of coexisting with.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Mami Inoue, 960-144, Okami-cho, Ushiku-shi, Ibaraki Prefecture (72) Kazuya Nakagome, 1-3-1, Higashi, Tsukuba-shi, Ibaraki Industrial Technology Institute ) Inventor Masahiro Asada 1-3-1 Higashi, Tsukuba-shi, Ibaraki Industrial Technology Institute of Biotechnology, Institute of Biotechnology (72) Inventor Makiko Sugie 1-3-1 Higashi, Tsukuba-shi, Ibaraki Institute of Industrial Science and Technology (72) ) Inventor Noboru Tozuka, 1-3 1-3 Higashi, Tsukuba-shi, Ibaraki Institute of Industrial Technology, Institute of Biotechnology, Institute of Biotechnology (72) Inventor, Yasunori Fukumori 1-3, Kita 4 West, Chuo-ku, Sapporo, Hokkaido Hokuren Agricultural Cooperative Federation

Claims (8)

[Claims] 1. An anti-allergic substance, which is obtained from a perilla hot water extract, has a mast cell degranulation inhibitory activity, and has a molecular weight of 13,500 and contains sugar and amino acid as constituent elements. 2. An anti-allergic substance, which is obtained from a perilla hot water extract and is a polyphenol having mast cell degranulation inhibitory activity. 3. The antiallergic substance according to claim 2, wherein the polyphenols are eluted with 50% acetone by DEAE anion column chromatography. 4. The anti-allergic substance according to claim 2, wherein the polyphenols are eluted with 50% acetone containing 0.1N HCl by DEAE anion column chromatography. 5. Extracting perilla leaves with hot water to separate solid-liquid,
The obtained aqueous solution is precipitated with a precipitant, the water-soluble component is eluted from the obtained precipitate, and the obtained eluted component is fractionated and purified by one or more column chromatography to remove mast cells. A method for producing an anti-allergic substance, which comprises obtaining a substance having a granule-inhibiting activity. 6. After separating and purifying by the column chromatography to obtain substances having mast cell degranulation inhibitory activity, each of the obtained substances is purified by affinity chromatography using a carrier on which hyaluronidase is immobilized. The method for producing an anti-allergic substance according to claim 5, wherein 7. An antiallergic agent comprising the antiallergic substance according to claim 1, 2, 3 and / or 4. 8. A functional food comprising the antiallergic substance according to claim 1, 2, 3 and / or 4 added to a food to impart an antiallergic function.