JPH08333267A - Antiallergic agent - Google Patents

Abstract

PURPOSE: To obtain an antiallergic agent derived from natural matter with its safety confirmed, thus optionally incorporable in medicines, cosmetics, foods/ beverages, etc. CONSTITUTION: A kind of Perilla frutescens crispa selected from rosemary, thyme and melissa is subjected to extraction with water or its mixture with a hydrophilic organic solvent to obtain a hyaluronidase-inhibitory extract which is used as the objective antiallergic agent.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antiallergic agent which can be used in various fields such as pharmaceuticals, cosmetics and foods.

[0002]

2. Description of the Related Art Hitherto, as antiallergic agents, histamine release inhibitors and competitive antagonists to histamine, such as chlorpheniramine maleate, diphenhydramine and related substances, have been proposed and used. In recent years, tranilast, sodium cromoglycate, baicalen, etc. have been reported as inhibitors of the release of inflammatory substances such as histamine and serotonin from mast cells that accompany type I allergic reactions. Although these anti-allergic agents exert excellent effects by selectively acting on the site of action, they often have side effects, and there is a problem in that their use and use conditions are severely limited.

[0003]

The object of the present invention is to use, as an anti-allergic agent, substances derived from natural products which have been used in ancient fields such as food and folk medicine and whose safety has been confirmed. The present invention is to provide an antiallergic agent that can be optionally mixed with medicines, cosmetics, foods and drinks.

[0004]

The present invention is obtained by subjecting a Lamiaceae plant selected from the group consisting of rosemary, thyme and melissa to extraction treatment with water or a mixture of water and a hydrophilic organic solvent. Based on the novel finding by the present inventors that the extract exhibits a strong hyaluronidase inhibitory action, the present invention provides an antiallergic agent containing the hyaluronidase alienating substance contained in the extract as an active ingredient. is there.

[0005]

[Function] Since an extract obtained from a natural product such as a plant has a complicated composition, when it exhibits an anti-allergic action, the components involved in the anti-allergic action are not single, and individual active ingredients Often the site of action is different. Therefore, in evaluating the anti-allergic action of natural products, it is desirable to perform not only a single evaluation method but also comprehensive evaluation from various viewpoints. Conventionally, there are several actions that are used as indicators of anti-allergic action, but the representative ones are the action of inhibiting the release of proinflammatory substances such as histamine from mast cells, the action of inhibiting hyaluronidase, and the action of inhibiting platelet aggregation. Therefore, a test using an animal allergy model is also conducted.

Hyaluronidase is strongly involved not only in maintaining the level of hyaluronic acid in the human body but also in the development of allergies. That is, when cells such as autologous cells appear due to an immune response to an allergen, the hyaluronidase possessed by these cells disrupts the tissue of the cell membrane and promotes tissue permeability and diffusion of substances inside and outside the cell, resulting in allergic symptoms. . Hyaluronidase is also present in mast cells and is activated when the IgE-antigen complex binds to the receptor, which promotes histamine release and exacerbates allergies. It has already been confirmed that many allergic symptoms are improved by suppressing the action of hyaluronidase.

An extract obtained by using a Lamiaceae plant selected from the group consisting of rosemary, thyme and melissa as water or a mixture of water and a hydrophilic organic solvent as an extraction solvent exhibits an excellent hyaluronidase inhibitory action, Furthermore, since it also exhibits a platelet aggregation inhibitory action and a hexosaminidase release inhibitory action (which serves as an index of histamine release inhibitory action), it is effective for the treatment of many allergic symptoms.

Next, a method for producing the antiallergic agent according to the present invention will be described. Rosemary (Rosmarinus officin
alis L.) is a plant of the Lamiaceae genus Mannenou and is also called Mannen wax. It is used as a flavoring agent for cooking, and is also known as a folk medicine for fever, sweating, stomach upset, etc.

Thym (Thymus vulgaris L.) is a plant of the family Lamiaceae, belonging to the genus Scutellariae, and is also called Tachimuskouso. There is. Melissa
ssa officinalis L.) is a plant of the family Lamiaceae, genus Astragalus genus.
It is also called Melissa Saw and is used as a raw material for the production of flavors for foods and cosmetics. In Europe, the exudate is said to be effective against sweating, headache, toothache and the like.

These rosemary, thyme or melissa can utilize the whole grass or above-ground part as an extraction raw material. Suitable extraction solvents include water, or water and hydrophilic organic solvents (eg methanol, ethanol,
Acetone, glycerin, 1,3-butylene glycol, etc.). Among them, water, water / ethanol mixture (ethanol content up to about 50%) and the like are preferable.

The extraction of the active ingredient is carried out by extracting the raw materials for extraction in a weight ratio of 5
It may be immersed in an extraction solvent of about -15 times at room temperature or under reflux heating. The extract obtained by distilling off the solvent from the extract, as it is, or even just subjected to a simple decolorization, can be used as an anti-allergic agent of the present invention,
It goes without saying that it may be subjected to any purification treatment effective for improving the hyaluronidase inhibitory activity and then used as an antiallergic agent.

As an active ingredient having an inhibitory effect on hyaluronidase in the extract of rosemary, thyme or melissa, carnosol which is a diterpene compound in the rosemary extract, and eriodisitol and biphenyl compounds which are flavonoid compounds in thyme are exemplified.
3,4,3 ', 4'-Tetrahydroxy-5,5'-diisopropyl-
Although 2,2'-dimethylbiphenyl and the like have been confirmed, it is speculated that some other components are involved in the hyaluronidase inhibitory activity.

The antiallergic agent of the present invention may be a hyaluronidase inhibitor extracted from either rosemary, thyme or melissa, a mixture thereof, or a mixture with another antiallergic agent.

The antiallergic agent of the present invention can be used as it is as a drug or a quasi drug, or can be blended with any cosmetics, foods and drinks, applied to the skin at the time of makeup, or ingested through daily eating and drinking. It may be done. Skin cosmetics such as lotion, cream, emulsion, shampoo,
When mixed with baby powder, rinse, hair tonic, soap, bath agent, pack, foundation, lip balm, lipstick, sanitary paper cotton (gauze, tissue paper, wet tissue paper, etc.), cosmetics with anti-allergic effect can be obtained. Provided.

When compounded in a cosmetic composition, the preferred amount of the antiallergic agent of the present invention is about 0.01 to 10% by weight in the case of a crude extract having an average hyaluronidase inhibitory activity. .

[0016]

【Example】

Example 1 50 g of dried whole grass of rosemary, thyme or melissa was subjected to extraction treatment with 500 ml of extraction solvent for 2 hours, and the obtained extract was concentrated to dryness to obtain an extract.

Table 1 shows the results of the above extraction treatment using various extraction solvents. (Extraction is 90 ° C for water,
In other cases, the heating was performed under reflux. The mixing ratio of water and alcohol is a volume ratio. )

[0018]

[Table 1] Extract yield (% by weight)  Extraction raw material Extraction solvent Rosemary thyme Melissa Water 16.1 26.7 25.4 Water / ethanol (1/1) 15.1 22.2 30.7 Ethanol 20.1 24.2 12.6 Water / 1,3-butylene glycol (1/1) 20.5 25.3 22.4

With respect to each of the obtained extracts, the hyaluronidase inhibitory action was examined by the following method. Hyaluronidase inhibitory activity test method: 20 μl sample solution and 0.18 ml buffer
And 0.1 ml of hyaluronidase solution are mixed and incubated at 37 ° C. for 20 minutes. Then 0.2 m of activator solution
and incubate at 37 ° C for 20 minutes. Add 0.5 ml of potassium hyaluronate solution to this and mix at 37 ° C for 4
After reacting for 0 minute, the reaction is stopped by adding 0.2 ml of 0.4N-NaOH aqueous solution. After cooling in ice, 0.2 ml of boric acid solution was added to the reaction mixture, boiled for 3 minutes and then cooled, p-D
After adding 6 ml of ABA reagent and incubating at 37 ° C. for 20 minutes, the absorbance A at 585 nm is measured. Apart from
Absorbance B was measured in the same manner as above except that no enzyme solution was added, and the absorbance C was measured in the same manner as above for the solvent used for sample dissolution. Absorbance D is measured in the same manner except that the solution is not added. From the measured absorbances A to D, the inhibition rate is calculated by the following formula. Inhibition rate (%) = [(CD)-(AB)] × 100
/ (C-D)

The above inhibition rate was measured by changing the concentration of the sample solution stepwise, and the 50% inhibitory concentration, that is, the inhibition rate was 50%.
The sample concentration is calculated by the interpolation method. Table 2 shows the test results
It was as follows. The hyaluronidase inhibitory activity of the extract obtained using only alcohol was inferior to that of the extract obtained using water or hydrous alcohol.

[0021]

[Table 2] Extraction material Extraction solvent 50% Inhibitory concentration (ppm) Rosemary water 122 〃 Water / ethanol (1/1) 115 〃 Water / 1,3-butylene glycol (1/1) 220 〃 Ethanol 380 thyme water 104 〃 Water / Ethanol (1/1) 148 〃 Water / 1,3-butylene glycol (1/1) 120 〃 Ethanol 246 Melissa water 62 〃 Water / ethanol (1/1) 92 〃 Water / 1,3-butylene glycol (1/1) 95〃 ethanol 296

Example 2 The rosemary water extract, thyme water extract, melissa water extract, rosemary ethanol extract, thyme ethanol extract and melissa ethanol extract obtained in Example 1 were prepared by the following method. The inhibitory effect on platelet aggregation was investigated.

Preparation of washed platelet suspension: 1/10 volume of 77 mM-EDTA was added to the blood of Japanese white rabbit and the rpm was adjusted to 1000 rpm.
Centrifuge for 10 minutes to remove the precipitate. 2100 rpm for the supernatant
Centrifuge at 10 minutes and collect the precipitated platelets. Float the obtained platelets in the platelet washing solution and rotate at 2100 rpm for 1
Centrifuge for 0 minutes. The precipitated platelets are collected and suspended in a platelet suspension so that the number of platelets will be 300,000 / μl.

Test method: 1 μl of a CaCl ₂ solution is added to 223 μl of the washed platelet suspension obtained by the above method, and the mixture is kept at 37 ° C. for 1 minute. 1 μl of the sample solution is added thereto, and the mixture is kept at the same temperature for 2 minutes, and then stirred for 1 minute. Next, add 25 μl of collagen solution, and after 10 minutes at 37 ° C,
The visible light transmittance A is measured and used as an index of the platelet aggregation state. Separately, the visible light transmittance B is measured in the same manner as above except that the sample solution is not added. The platelet aggregation inhibition rate is calculated from the measured visible light transmittance by the following formula. Platelet aggregation inhibition rate (%) = [(B−A) / B] × 100

The concentration of the sample solution is changed stepwise to measure the above platelet aggregation inhibition rate, and the 50% inhibition concentration, that is, the concentration of the sample solution at which the platelet aggregation inhibition rate becomes 50% is determined by the interpolation method. The test results are shown in Table 3. The inhibitory effect on platelet aggregation of the extract obtained using only alcohol was inferior to that of the extract obtained using water or hydrous alcohol.

[0026]

[Table 3] Extraction material Extraction solvent 50% Inhibitory concentration (ppm) Rosemary Water 325〃 Water / Ethanol (1/1) 280〃 Water / 1,3-Butylene glycol (1/1) 750〃 Ethanol Measured with weak activity Impossible time water 178 〃 water / ethanol (1/1) 190 〃 water / 1,3-butylene glycol (1/1) 360 〃 ethanol 330 melissa water 295 〃 water / ethanol (1/1) 320 〃 water / 1, 3-Butylene glycol (1/1) 1050〃 Ethanol Not active due to weak activity

Example 3 With respect to the rosemary water extract, thyme water extract and melissa water extract obtained in Example 1, histamine release inhibiting action was carried out by the following method using the hexosaminidase release inhibiting rate as an index. I checked.

Test method: 1.0 × 10 ⁶ RBL-2H3 cells were placed in a 25 ml flask and MEM with 15% FBS was added.
The medium is inoculated and cultured at 37 ° C. under 5% CO ₂ for 4 days, and then trypsinized and centrifuged (1000 rpm, 2 minutes) to obtain a cell fraction as a precipitate. This was suspended in the same medium as the above medium at 4.0 × 10 ⁵ cells / ml, and 5 μl of mouse monoclonal anti-dinitrophenyl group IgE (DNP-specific IgE) was added thereto to a concentration of 0.5 μg / ml. To do. 96 wellsplat the obtained cell suspension
Inoculate 80 μl into e and incubate at 37 ° C. under 5% CO ₂ for 24 hours. After culturing, remove the medium from each well and remove Siragani
Wash with an buffer (500 μl x 2). Next, 30 μl of the above buffer solution is newly added, 10 μl of the sample solution is further added, and the mixture is incubated at 37 ° C. for 10 minutes. Then dinitrophenylated bovine serum albumin (DNP-BSA)
Add 10 μl of solution and incubate again at 37 ° C. for 10 minutes. Then, add 10 μl of the supernatant to a new 96wel under ice cooling.
Transfer to ls plate and add 1mM p-nitrophenyl-
Add 10 μl of N-acetyl-β-D-glucosamide solution and add 3
Incubate at 7 ° C for 1 hour. 0.1 after completion of reaction
Add 250 μl of M Na ₂ CO ₃ NaHCO ₃ buffer and measure the absorbance A at 415 nm with a microplate reader. The same process and the absorbance measurement are performed on the cell suspension to which the sample solution is not added (the absorbance measured at this time is B). Also, the same treatment and absorbance measurement are performed using the above buffer solution instead of the cell suspension (the absorbance measured at this time is C). Then, the hexosaminidase release inhibition rate is calculated by the following formula.

Hexosaminidase release inhibition rate (%) =
[1- (AC) / (BC)] × 100 Separately, a test using baicalin as a positive control was also performed. The test results are shown in Table 4, and it was confirmed that the rosemary water extract, the thyme water extract, and the melissa water extract showed a stronger activity than baicalin which is a known active substance, and had a remarkable inhibitory effect on histamine release. Was done.

[0030]

Table 4 percent inhibition of the specimen 25ppm (%) rosemary water extract 18.4 time water extract 31.6 Melissa water extract 26.2 baicalin (positive control) 13.5

Example 4 The antiallergic effect of the rosemary water extract, the thyme water extract and the melissa water extract according to Example 1 was examined by using the type IV allergy suppressing action by the contact dermatitis model as an index. The test method is as follows.

Test method: ddy preliminarily kept for 1 week after purchase
Abdomen of male mouse strain (8 weeks old; 5 mice per group) 25 x 20 mm
Shave the range of hair with a razor. The next day, 100 μl of sensitizing antigen (7% picryl chloride / ethanol solution) was added.
Drip and apply evenly. After 6 days, the thickness of the right auricle is measured, and then 20 μl of a challenge antigen (1% picryl chloride / olive oil solution) is applied to the front and back of the right auricle for primary induction. The thickness of the right auricle is measured 24 hours after the application. Next, a mouse in which the swelling degree of the right auricle due to the above-mentioned temporary induction was high (the sensitization was sufficiently established by the primary induction) was selected, and the sample solution (1% aqueous solution) was applied to the front and back of the right auricle daily. After continuously applying 20 μl for 1 week, 100 μl of the same sensitizing antigen as described above is again dropped and applied to the abdominal shaving part to perform sensitization. Then, 20 μl of the sample solution is applied to the front and back of the right auricle daily for 1 week continuously, and then the thickness d of the right auricle is measured. After that, 20 μl of the challenge antigen was applied to the front and back of the right auricle to induce it, and after 24 hours, the thickness D of the right auricle was measured, and the edema rate [(D−d) × 1 was obtained from the measured value.
00 / d]. Water is applied instead of the sample solution to the control group, and the same measurement is performed. From the measurement result, the suppression rate is calculated by the following formula.

Inhibition rate (%) = [(A−B) / A] × 100 (A: edema rate of control group; B: edema rate of sample solution treated group) The test results are shown in Table 5.

[0034]

[Table 5] Edema rate (%) Suppression rate (%) Control group 101.9 ± 6.04 − Rosemary extract treatment group 65.7 ± 4.20 35.5 * Thyme extract treatment group 69.7 ± 1.65 31.6 * Melissa extract treatment group 85.9 ± 4.46 15.8 * (*: risk rate 1% or less)

[0035]

Industrial Applicability As described above, the antiallergic agent of the present invention is not only excellent in antiallergic action, but also contains as an active ingredient a plant extract which has been used in the fields of food and cosmetics for a long time. Since it is a thing, it is also excellent in safety.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shizuko Kataoka 14703-10 Muto-cho, Onomichi, Hiroshima Prefecture Maruzen Pharmaceutical Co., Ltd. (72) Kenji Mizutani 14703-10, Muto-cho, Onomichi, Hiroshima Prefecture Maruzen Pharmaceutical Co., Ltd. Within

Claims (3)

[Claims] 1. An anti-allergic agent comprising as an active ingredient a hyaluronidase inhibitor extracted with water from a Lamiaceae plant selected from the group consisting of rosemary, thyme and melissa. 2. A hyaluronidase inhibitory extract obtained by subjecting a Lamiaceae plant selected from the group consisting of rosemary, thyme and melissa to extraction treatment with water or a mixture of water and a hydrophilic organic solvent. Anti-allergic agent. 3. An anti-allergic agent comprising an extract obtained by extracting a Lamiaceae plant selected from the group consisting of rosemary, thyme and melissa with water or a mixture of water and ethanol.