JP2946183B2 - Perilla extract having TNF production inhibitory action - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel extract which is effective for suppressing TNF production from a Labiatae plant as a raw material. More specifically, the present invention relates to a novel extract that is effective for improving allergy such as atopic dermatitis by suppressing TNF production.

[0002]

BACKGROUND OF THE INVENTION Many allergic reactions are involved in the onset of cedar pollen, chile, rhinitis caused by animal hair, mites, urticaria caused by milk, etc., and atopic dermatitis. Allergy is due to a reaction between an antigen and an antibody in a living body. The reaction in the living body may occur weakly or strongly, immunity occurs weakly, and hypersensitivity occurs strongly. Hypersensitivity is roughly classified into immediate type hypersensitivity and delayed type hypersensitivity, and is classified into types I to IV according to the onset mechanism. The allergies currently in question are overwhelmingly type I.

[0003] In general, an allergic reaction is a reaction of an antigen-antibody reaction in a living body, resulting in histamine, acetylcholine, bradykinin, SRS which are chemical mediators of the living body.
-A (anaphylaxis slow-reacting substance) and the like are released, and this is interpreted as a biological reaction caused by damaging the tissue. Atopic dermatitis, a type of allergic reaction, refers to congenital hypersensitivity to certain substances specific to humans, especially children, and further develops as bronchial asthma, allergic rhinitis, and hay fever. It is said that the predisposition is hereditary and is characterized by a high degree of hypersensitivity to food and absorbed antigens.

[0004] Atopic dermatitis is a dermatitis caused by such a predisposition, and its subjective symptom is a remarkable pruritus, and it is said that the eruption is hatred and chronic by scratching. . Conventionally, as a treatment method for allergic diseases represented by such atopic dermatitis,
There are systemic therapies with oral medications such as antiallergic agents, antihistamines and steroids.

[0005] However, since these internal medicines are medical medicines, a doctor's diagnosis and instructions are required at the time of prescription and treatment. In addition, there is a steroid ointment containing a corticosteroid as a topical treatment, but it may cause an acute disease, and has a drawback that the percutaneous absorption adversely affects the endocrine system. On the other hand, Lamiaceae plants are most characterized by their aroma, and its main component is known to be perylaldehyde, which is used as a fragrance. On the other hand, perillaldehyde is known to cause allergic contact dermatitis in perilla growers (Dermatology: 3 (8), 713-176, 1981 and skin: 24 (2), 250-25
6,1983).

[0006]

BACKGROUND OF THE INVENTION Studies on inflammation and allergic reactions have revealed that cytokines emerge from leukocytes and act against antigen invasion. Tumor necrosis factor (hereinafter referred to as T), which is a kind of such cytokines, is known.
Abbreviated as NF. ) Was recently found to be very directly related (Bonavida B. Biotherapy, 3, P1
27 (1991)). Therefore, the present inventors thought that suppressing the amount of TNF production would suppress allergic inflammation such as atopic dermatitis, and conducted intensive research. As a result, it has been found that the perilla extract has an inhibitory effect on TNF production, and the inhibitory effect is improved by removing components in the high molecular weight region of 10,000 or more. At the same time, by removing the high molecular weight region of 10,000 or more, the coloring degree of the extract was reduced, and it was confirmed that it was convenient for commercialization into foods and cosmetics.

[0007]

2. Description of the Related Art Conventionally, as perilla extract, perilla extract (Alps Pharmaceutical Co., Ltd.) and perilla extract described in Permissible Raw Material Standards for Cosmetics Varieties have been generally distributed. Many patents relating to perilla extract have also been reported (Japanese Unexamined Patent Publication No.
-86516, 50-6750, 55-81896, and 57-53403), and the inventors of the present application also applied for a patent on a food made from a Labiatae plant (JP-A-4-79852). These contain perylaldehyde, which is an aroma component, or contain components having a molecular weight of 10,000 or more.

On the other hand, it has been disclosed that rosmarinic acid (molecular weight: 360) contained in a Labiatae plant is effective as a 5-lipoxygenase action inhibitor and can be used as an anti-allergic agent or an anti-allergic food (Japanese Unexamined Patent Publication (Kokai)). 56-99412, JP-A-1-121217, etc.). However, when the present inventors measured the TNF production inhibitory effect of the rosmarinic acid fraction separated from the perilla extract of the present invention in vitro, the TNF production inhibitory effect was not recognized, and the rosmarinic acid fraction was not observed. Improvement of atopic dermatitis was not observed in cosmetics (cream) containing the acid fraction (see Test Examples described later).

[0009]

DISCLOSURE OF THE INVENTION In order to solve the conventional drawbacks, the present inventors have conducted intensive studies on Labiatae plants and their closely related species, and obtained from extracts of Labiatae plants having a molecular weight of 10,000 or more. It has been found that an extract obtained by selectively removing fractions and that does not contain perylaldehyde specifically suppresses TNF production, and that these perilla extracts are useful for allergic diseases such as atopic dermatitis. After confirming that there is a great effect, the present invention has been completed.

[0010]

That is, the present invention provides a method for producing perillaldehyde and a compound having a molecular weight of 10,000 or more from components obtained by grinding foliage of a Labiatae plant and extracting with water, an organic solvent or a mixture thereof. The present invention provides a perilla extract having a TNF production inhibitory effect and a low degree of coloration, which is obtained by removing the fraction.

[0011] Perilla used in the present invention is a scientific name of Perilla flutesc-ens Britton var.cr.
ispa DENSE and var.acuta Kudo), and other related plants (Labiatae). Originated in East India and China,
It is widely cultivated in the temperate regions of the Orient. In the present invention, blue sesame, blue crepe mess, red crepe, crepe mess, kame mess, etc. produced in Japan can be preferably used.

As the extraction solvent, water, an organic solvent and a mixture thereof are used. Examples of the organic solvent include alcohols such as methanol, ethanol, isopropanol and propylene glycol, acetone and chloroform. And aqueous ethanol solutions are preferred. At this time, perylaldehyde in the extract is removed by a filtration treatment (eg, activated carbon). Methods for removing the fraction having a molecular weight of 10,000 or more include ultrafiltration, addition of a hydrophilic organic solvent, addition of an ammonium salt or an inorganic metal salt,
Perform gel filtration.

The extraction process is generally performed as follows.
That is, the stems and leaves of perilla are ground, added to a solvent, and heated with stirring at room temperature to 50 ° C. for 1 to 10 hours. Then, the mixture was filtered under reduced pressure, deionized water was added to the filtrate, a small amount of protease was added, and the mixture was allowed to stand at a temperature of about room temperature to about 40 ° C for 1 to 24 hours with occasional stirring. Excluding 10,000 or more fractions. Thereafter, the mixture is concentrated under reduced pressure, filtered through an activated carbon layer, and the filtrate is sterilized by heating to obtain a perilla extract.

The extract of the present invention may be used alone or as a raw material in the form of powder obtained by spray-drying, freeze-drying, granulation drying, etc., as an antiallergic food, health food, cosmetics, quasi-drug, or other forms. Can be used with In order to make it an anti-allergic food, it is sufficient that the extract is usually in any food form that allows the user to eat the extract, for example, the extract alone, or soft drinks, confectionery, bread, noodles, batter products, tea, Examples include dressing, candy, candy, gum and the like.

When used as a health food, for example, it may be used alone or powdered by syrup, liquid, spray-drying or freeze-drying, and then mixed with a carrier or excipient by a conventional method to give tablets, It can be used in the form of preparations such as sugar coatings, powders, capsules, dry syrups, granules and the like. In addition, cosmetics and quasi-drugs include, for example, ointments, liniments, aerosols, creams, soaps, facial cleansers, whole body cleansers, shampoos, rinses,
Treatments, hair styling agents, hair tonics, hair restorers, lotions, lotions, make-up cosmetics, packs, baby powders, bath salts, shipping agents, lotions impregnated in roll paper manufactured from pulp, non-woven fabric, and the like. .
Furthermore, in order to impregnate with a perilla extract or the like to make a fiber product, it may be in any form directly in contact with the user,
Examples include towels, underwear, socks and the like.

[0016]

The present invention will be described below with reference to production examples and test examples of the antiallergic perilla extract of the present invention.

[Production example] 100 g of ground chili mendiso
Then, 400 ml of a 40% ethanol solution was added thereto, followed by stirring and extraction at 40 ° C. for 4 hours. It was filtered under reduced pressure, 5 liters of deionized water was added to the filtrate, a small amount of protease was added, and the mixture was allowed to stand for 12 hours at 40 ° C. with occasional stirring, and was then subjected to an ultrafiltration membrane (Advantech Ultrafilter Q100, (Molecular weight of 10,000) The fraction having a molecular weight of 10,000 or more was removed. The permeate from the ultrafiltration was concentrated under reduced pressure to about 150 ml, followed by activated carbon filtration. 3 filtrates
Heat sterilized at 90 ° C for 0 minutes, and after cooling, 1
00 g of perilla extract was obtained.

Properties of perilla extract The extract described above contains protein (by Kjeldahl method), lipid (by ether extraction method), ash (by dry ashing method), moisture (by normal pressure heating and drying method) and carbohydrate (by atmospheric heating drying method). Table 1 shows the results of measuring the contents of the respective components (calculated as the residual amounts of the respective components).

[0019]

[Table 1]

Detection of perillaldehyde in perilla extract The perilla extract of the present invention (A) and perilla extract (B) (manufactured by Maruzen Pharmaceutical Co., Ltd.) based on permissible raw materials according to cosmetic varieties were analyzed by gas chromatography under the following conditions. Aldehyde analysis was performed. Column: Capillary column (TC-WAX, 60mx
0.25 mm, I.V. D. 0.25 μl) Detector: FID Moving layer gas: helium Temperature: 220 ° C. → 230 ° C. (heating: 1 ° C./min) Sample amount: Each sample was diluted 5-fold with ethanol and the same amount was injected into the column. The obtained gas chromatogram is shown in FIG. That is, it can be seen that the perilla extract of the present invention does not contain perylaldehyde.

Visible Absorbance of Perilla Extract in Perilla The above-mentioned perilla extract of the present invention and the perilla extract without the ultrafiltration membrane were each diluted 10 times (w / w) with deionized water, The visible absorbance was measured with a spectrophotometer U-2000 (optical path length 1 cm). Table 2 shows the results. That is, it can be seen that the degree of coloring was reduced by ultrafiltration.

[0022]

[Table 2]

[Test Examples] The effects on the immunosuppressive function will be described with reference to the following test examples. (1) Tumor necrosis factor (TN) in vivo
F) Production inhibitory action Orally administered 0.4 g of perilla extract sample to mice,
After a lapse of time, an immunostimulant (OK432: Picibanil, manufactured by Chugai Pharmaceutical Co., Ltd.) was administered. Blood was collected 2 hours later, and TNF activity was measured. Table 3 shows sample names and measurement results.

[0024]

[Table 3]

As is evident from Table 3, the amount of TNF produced by the perilla extract administration group was reduced to about 2/3, but further reduced to 1/3 by ultrafiltration, and the immunosuppressive action was enhanced. It has been shown.

(2) TN in vitro
F production inhibitory action Macrophages collected from the mouse peritoneal cavity were added to the test sample solution, incubated at 37 ° C for 1 hour, and stimulated with LPS (Lipopolysaccaharide).
After time, TNF activity was measured. As a test sample,
A control (distilled water), a perilla extract (product of the present invention) and a rosmarinic acid fraction were used. The rosmarinic acid fraction is obtained from the perilla extract of the present invention by using an open column of styrene-divinylbenzene-based porous polymer carrier (eluent: water-methanol).
, Dried under reduced pressure, and added with an equal amount of deionized water to the original extract. Table 4 shows the measurement results.

[0027]

[Table 4]

From Table 4, it is clear that the perilla extract has an effect of suppressing TNF production, and the effect is not due to rosmarinic acid.

(3) Administration test Seven subjects (A to G) with atopic dermatitis were instructed to drink 0.5 g of the perilla extract of the present invention every day and judged 10 days later. Table 4 shows the test results.

[0030]

[Table 5]

From the above, it was clarified that drinking the perilla extract was effective for atopic dermatitis.

(4) Formulation test for cream and use test (i) Formulation test for cream A cream prepared by mixing the perilla extract of the present invention with the composition shown in Table 6 was prepared. A cream having no problem in terms of viscosity was obtained.

[0033]

[Table 6]

(Ii) Cream use test Creams containing perilla extract shown in Table 6 above (a: 35 subjects) were tested on 35 subjects with atopic dermatitis, respectively.
Rosmarinic acid fraction-containing cream (c: 10 subjects)
And a control cream (d: 35 subjects) was applied to the affected area every day, and four weeks later, it was judged according to the following criteria. ○: Remarkably improved (pruritus, erythema, desquamation, infiltration almost disappeared) △: Slightly improved (pruritus, erythema, desquamation, infiltration was slightly reduced) ×: unchanged (pruritus, erythema, desquamation, infiltration unchanged) The test results are summarized in Table 7.

[0035]

[Table 7]

The results in Table 7 show that the cream containing the perilla extract is effective against atopic dermatitis such as pruritus,
It can be seen that the effect is not due to rosmarinic acid.

[Brief description of the drawings]

FIG. 1 is a gas chromatogram of a perilla extract (A) of the present invention, a perilla extract (B) based on a licensed raw material standard for each cosmetic variety, and perylaldehyde as a standard.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI A61K 31/00 643 A61K 31/00 643D C07G 17/00 C07G 17/00 Z // C11B 9/00 C11B 9/00 Z (56) References JP-A-4-79852 (JP, A) JP-A-60-32711 (JP, A) Biosci. , Biotechnol. , Biochem. , 56 (1) (1992). p. 152 (58) Field surveyed (Int.Cl. ⁶ , DB name) A61K 35/78 A23L 1/30 A61K 7/00 A61K 7/48 C07G 17/00 C11B 9/00

Claims (6)

(57) [Claims] 1. A TNF obtained by grinding the foliage of a Labiatae plant and extracting it with water, an organic solvent or a mixture thereof to remove perylaldehyde and a fraction having a molecular weight of 10,000 or more. A perilla extract with a low degree of coloration, which has a production inhibiting effect. 2. The perilla extract according to claim 1, which has been subjected to an extraction treatment using an aqueous ethanol solution. 3. The perilla extract according to claim 1, wherein perillaldehyde is removed by activated carbon filtration. 4. The perilla extract according to claim 1, wherein a fraction having a molecular weight of 10,000 or more is removed by ultrafiltration. 5. The stem and leaves of a Labiatae plant are ground, extracted with an aqueous ethanol solution at room temperature to 50 ° C., filtered, added with a protease and stirred, and then subjected to ultrafiltration with a molecular weight of 10,000. The perilla extract according to claim 1, wherein the above fractions are removed and perillaldehyde is further removed through an activated carbon layer. 6. The perilla extract according to claim 1, which has an atopic dermatitis reducing effect.