KR20090130021A - Anti-histamine substance and method for production thereof - Google Patents

Abstract

Disclosed is a method for producing an anti-histamine substance, which comprises the steps of: adding an yeast cell and water to a driedplant tissue and causing the fermentation of the mixture; extracting a fermentedproduct produced in the fermentation step with an aqueous ethanol solution; concentrating an extract produced in the extraction step; and fractionating a concentrated solution produced in the concentration step and collecting a 90% ethanol-soluble fraction.

Description

Anti-histamine substance and method for production thereof

The present invention relates to an allergic symptom improving substance that causes pollen, urticaria, bronchial asthma, atopic disease and the like and a method for producing the same.

An allergy means that an immune response occurs in excess for a particular antigen. Allergies are largely classified into type I to V depending on the mechanism of their occurrence. Pollenosis, gallbladder, and bronchial asthma are classified as type I. In addition, atopic disease is said to be close to type I allergy. Type I allergy is thought to occur because IgE antibodies bind to mast cells or basophils, resulting in the release of histamine and leukotrienes released from these cells throughout the body.

It is known that a fermentation concentrate of stevia stem is used as an active ingredient as an external dermatosis therapeutic agent effective for atopic dermatitis or the like or as an internal medicine for treating gastrointestinal diseases such as gastritis and gastric ulcer. It is also known that fermentation of plant tissue extract of Stevia is known as a substance having an antihistamine effect effective for allergic symptoms such as hay fever.

In addition, in view of the problem that the above technique requires a long period of 90 days or more for stevia plant tissue fermentation, the present inventors can shorten the period required for fermentation of stevia extract, thereby obtaining a substance for improving allergic symptoms. The plant tissue of Stevia was ground, mixed and put into purified water to obtain a filtrate by heating, stirring, cooling, and filtration. The filtrate was concentrated under reduced pressure to obtain Stevia X, and the yeast was added to the Stevia X to ferment. A method for producing an allergic symptom improving substance was previously proposed (see Japanese Patent Laid-Open No. 2005-35888).

As described above, many substances for improvement such as hay fever and atopic disease have already been proposed and marketed. However, many diseases are commonly desired to improve the degree of improvement of allergic symptoms. Among these situations, research and development are being vigorously pursued in search of an allergic substance improving substance than before.

An object of the present invention is to provide a substance that exhibits an antihistamine effect superior to that of the prior art, in particular, to help improve the symptoms of type I allergy.

In order to solve the above problems, the inventors of the present application succeeded in producing a substance exhibiting much superior antihistamine action using stevia plant tissue as a raw material. The method for producing an antihistamine substance according to the present invention having the above-mentioned effects comprises the steps of adding yeast and water to a dried stevia plant tissue, fermenting stevia obtained by the fermentation step using an aqueous ethanol solution, And extracting the 90% ethanol soluble fraction by fractionating the extract obtained by the extracting step and fractionating the concentrate obtained by the concentration step.

The inventors of the present invention have found that strong antihistamine action is recognized in a substance obtained through the above production method. Therefore, the substance can be widely applied to cosmetics, external preparations, oral preparations, etc., which are particularly suitable for people with type I allergy symptoms.

In addition, the material obtained by the above-described manufacturing method has no unpleasant smell. On the other hand, there existed a problem that the antihistamine substance obtained by the manufacturing method of the said patent document 1 may have an unpleasant smell. Since the substance obtained by this invention does not have such a problem, it has the advantage that it is easier to use than a conventional substance when mix | blending with external preparations, especially cosmetics. Therefore, this invention includes the cosmetics and pharmaceuticals (external agent, oral preparation) containing the substance obtained by the manufacturing method mentioned above in the range.

1 is a flow chart showing the flow of a method for producing an antihistamine material according to the present invention.

Figure 2a is a diagram showing one of the experimental results of examining the antihistamine effect of the present invention.

Figure 2b is a diagram showing one of the experimental results for examining the antihistamine effect of the present invention.

Figure 2c is a diagram showing one of the experimental results for examining the antihistamine effect of the present invention.

Figure 3 is a diagram showing one of the experimental results for examining the antihistamine effect of the present invention.

Detailed Description of the Preferred Embodiments

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail using an accompanying drawing.

First, a method for preparing an antihistamine material according to the present invention according to the flowchart of FIG. 1 will be described in detail.

Process 1 is a process of drying and grinding the plant structure of stevia used as a raw material. The stevia plant, which is one of the raw materials of the present invention, is a perennial plant originating in South America Paraguay, and its scientific name is called Stevia Rebaudiana Bertoni. When cultivating in Japan, it is common to start cultivating seed, rhizome or sesame seedlings from February to May. Harvesting is often carried out from October to early November when the stevia plants are sufficiently mature.

The antihistamine substance of this invention uses the dried stevia powder which made the stevia plant tissue dry well, and grind | pulverized it finely as a raw material. Even if it is powder, it is enough to grind | pulverize so that each piece may be about 1 cm or less in size. Moreover, it is preferable to select and use the leaf and stem part of a stevia plant.

It is preferable to perform the drying of the leaves and stems of the stevia plant at room temperature in a well-ventilated room, and it is not preferable to apply rapid heat.

Step 2 is a step of fermenting the dry stevia powder obtained in Step 1. As described above, the method for producing an antihistamine substance according to the present invention is characterized in that the dry stevia powder is directly fermented. In contrast, in the production method disclosed in Japanese Patent Laid-Open No. 2005-35888, the extract is first extracted from the dry stevia powder and the extract is fermented. However, according to the results of subsequent studies by the inventors of the present invention, it has been found that the fermented dry stevia powder can produce a substance having a strong antihistamine effect.

Fermentation of dry stevia powder can be performed by adding water and yeast, and stirring and leaving to dry stevia powder. The amount of water to be added may be any amount necessary for the fermentation, and the amount of the whole wet is sufficient. As yeast, it is preferable to use Saccharomyces.

Fermentation of the dry stevia powder is preferably carried out "completely." When you taste unfermented stevia powder, it tastes sweet, but the fermented stevia powder does not feel sweet. Therefore, it is preferable to proceed with the fermentation until no sweetness is felt at all. Full fermentation requires approximately two to three weeks of standing at room temperature.

The type of yeast uses Saccharomyces. In the fermentation process, first, the dried stevia powder is moistened, the yeast is added thereto, and the fermentation is continued while supplying moisture for 2 to 3 weeks at room temperature. The determination of whether or not the fermentation of the stevia powder has been "completely" can be performed, for example, by considering the end of the "complete" fermentation when the stevia powder is in the mouth and its sweetness is not felt.

Step 3 is a step of extracting and concentrating the fermented stevia powder obtained in Step 2, but it is important to use ethanol for extraction. This is because collecting 90% ethanol soluble fraction in the following process 4 is one of the features of the present invention.

Ethanol extraction can be performed by preparing about 30% of ethanol aqueous solution, adding fermentation stevia powder to this, and infiltrating at room temperature for several hours to several days, stirring slowly. The filtrate obtained by filtering this to the magnitude | size of about 140 mesh is used as an extract liquid. In the production method according to the present invention, the extract is concentrated under reduced pressure at a temperature of about 60 ° C.

Step 4 is a step of collecting the 90% ethanol soluble fraction by fractionating the concentrated extract obtained in step 3. Fractionation can be performed by passing a concentrated extract liquid through an ion exchange resin column such as DIAION® HP-20. Strong antihistamine action is recognized for the fractionated 90% ethanol soluble fraction.

The 90% ethanol soluble fraction fractionated for convenience of storage and transportation may be frozen and dried (step 5). This completes the process for producing the antihistamine substance in the preferred embodiment of the present invention.

<Test Example>

Pharmacological experiments on antihistamine activity were carried out using the morphot extraction ileum specimens of the present invention obtained through steps 1 to 5 shown in FIG. The experiment was carried out by extracting the ileum from morphot and measuring the isometric contraction induced by histamine by the Magnus method.

The application of histamine ^10-6 M (mortamine ^10-6 M) to the morphologic extraction ileum specimens causes contraction. Therefore, the effect of the present invention on the shrinkage reaction was investigated by applying histamine 10 ^-6 M before and after the application of the present invention obtained by the above-described method to observe the shrinkage reaction difference. Shrinkage was recorded with a direct current amplifier (Unipulse Inc. AM20) through a tension transducer (BG-10, Kulite Semiconductor, USA). Histamine was used by Wako Pure Chemical Industries, Ltd.

For the application of the material of the present invention, a test solution in which the material of the present invention was dissolved in a Lock-Ringer solution was prepared. Making a solution of the present invention a concentration of three different materials was adjusted to be the concentration of each respective 1O ^-5 g / ㎖ of the invention ^{substance, 10 -4 g / ㎖, 1O} -3 g / ㎖. The test solution was applied directly to the Magnus tube.

The composition of the Lock Ringer solution was NaCl = 154, KCl = 5.6, CaCl ₂ = 2.2, MgaCl ₂ = 2.1, NaHCO ₃ = 5.9, glucose = 2.8 (pH = 7.4). The nutrient solution was vented with a mixed gas of 95% O ₂ and 5% CO ₂ .

Experiments were conducted at room temperature (23-24 ° C.).

The experimental results are shown in Figs. 2A to 2C. Figure 2a, the material density of the present invention ¹⁰ is the experimental results for the ⁵ g / ㎖ of solution. At 6:22, 10 ^-6 moles of histamine was applied to the morphot extracted ileum specimens to measure the contractile response, which was called the control. After the shrinkage of the sample was completed, a sample of ^10-5 g / ml of the inventive substance concentration was applied to the sample at 6:30 and ^10-6 moles of histamine was again applied to the sample at 6:33. The recording graph by Unipulse Co., Ltd. AM20 in this measurement was as shown in FIG. 2A.

The abscissa of the graph of FIG. 2A represents time and represents two minutes in one mass. The vertical axis represents voltage and is approximately proportional to the size of the contraction of the ileum specimen (FIG. 2B and FIG. 2C as well). When the contraction size of the first histamine application was 100, the contraction size of the second histamine application was 89.7 on the graph. The decrease in contraction size during the second histamine application is thought to be due to the antihistamine effect of the test fluid.

Fig. 2B is a recording graph of Unipulse Co., Ltd. AM20 as a result of an experiment on a reagent having a concentration of 10 ^-4 g / ml of the present invention. At 4:47, 10 ^-6 moles of histamine was applied to the morphot extracted ileum specimens to measure the contractile response, which was called the control. After the shrinkage of the sample is completed, a 4:55 present invention concentrations of ^10-4 / ㎖ of the solution and applied to the sample, and the histamine 4:00 ^10-6 again after 58 minutes was applied to the sample in moles.

When the concentration of the present invention in the test solution was 10 ⁻⁴ / ml, when the shrinkage size (voltage value of the direct current amplifier) at the first histamine application was 100, the shrinkage size at the second histamine application was determined to be 60 on the graph. Could. Therefore, it can be seen that a strong antihistamine effect is exhibited compared to the case where the concentration of the substance of the present invention is 10 ⁻⁵ / ml.

Fig. 2C is a recording graph showing the results of experiments on a reagent having a concentration of 10 ^-3 g / ml of the present invention. At 6:40, 10 ^-6 moles of histamine is applied to the morphot extract ileum sample to measure the contractile response, which is called the control. After the shrinkage of the sample was completed, a sample of 10 ⁻³ / ml of the substance concentration of the present invention was applied to the sample at 6:50, and 10 ⁻⁶ mol of histamine was again applied to the sample at 6:52.

As can be seen in Figure 2c, when the concentration of the present material of the solution is 10 ^-3 / ㎖, there is no shrinkage reaction of the sample at the time of the second histamine application. This was a surprising result that was never expected even by the inventor of this application. In view of these results, it can be understood that the antihistamine effect of the substance of the present invention is a powerful force far beyond conventional common sense.

Fig. 3 is a graph of the experimental results shown in Figs. 2A to 2C, in which the horizontal axis represents the logarithm of the concentration of the substance of the present invention in the test solution, and the vertical axis represents the ratio of the measured value of the shrinkage reaction after application of the test solution and the control group. It is apparent that the shrinkage reaction decreases as the concentration of the substance of the present invention in the test solution increases.

As mentioned above, the detail of the manufacturing method of the antihistamine substance by this invention, and the experiment result which illuminates the effect of the antihistamine substance manufactured by the manufacturing method were shown. From the experimental results, it was shown that the antihistamine substance according to the present invention has a surprising antihistamine effect. Therefore, the antihistamine material according to the present invention can be widely applied to cosmetics and medicines, and in particular, it can be expected to be applied to cosmetics and medicines suitable for people with type I allergy such as hay fever or atopic disease or those showing near symptoms. Furthermore, the antihistamine material according to the present invention is characterized by not having an unpleasant smell at all, which makes it easy to apply to cosmetics sensitive to odor problems. Therefore, the antihistamine substance which concerns on this invention is anticipated very much as a component mix | blended with cosmetics for people who show symptoms, such as hay fever and atopic disease.

In the cosmetics, the antihistamine substance according to the present invention is cosmetics for hair, hairdressing, wool, scalp, hair coloring, hair shampoo, hair rinse, skin cosmetics / cosmetics, cosmetics, cream, milky lotion, sunscreen, sunscreen, washing Cosmetics, shaving agents, hair cleansers, facial rinses, packs, cosmetic oils, body rinses, massaging agents, cosmetics and foundations for finishing, makeup bases, powders, lipsticks, eye makeup, cheek cosmetics, body makeup In cosmetics such as cosmetics for baths, nail cosmetics, body powders, etc.In medicines, powders, granules, tablets, capsules, pills, liver preparations, pencils, internal medicines, external preparations, extracts, warnings, suppositories , Aerosol, gas agent, chemical adsorbent, ophthalmic solvent, injection, band-aid etc.

Claims (5)

As a method for producing an antihistamine substance, Fermentation by adding yeast and water to the dried stevia plant tissue, Extracting the fermentation stevia obtained by the fermentation step using an aqueous ethanol solution, Concentrating the extract obtained by the extraction step, and Collecting a 90% ethanol soluble fraction by fractionating the concentrate obtained by the concentration process; Manufacturing method comprising a. The method of claim 1, The fermentation process is characterized in that the stevia plant tissue is completely fermented. An antihistamine substance comprising 90% ethanol soluble fraction of a concentrate obtained by extracting fermented stevia obtained by adding yeast and water to the dried stevia plant tissue and fermenting with an aqueous ethanol solution, and concentrating the extract. An antihistamine substance produced by the production method according to claim 1 or 2, or a cosmetic comprising the antihistamine substance according to claim 3. A pharmaceutical comprising the antihistamine substance produced by the manufacturing method of claim 1 or 2, or the antihistamine substance according to claim 3.

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