WO2004002506A1

WO2004002506A1 - Agent for curing anaphlactic disease and health food, and manufacturing process thereof

Info

Publication number: WO2004002506A1
Application number: PCT/JP2003/008223
Authority: WO
Inventors: Koji Fukuda; Harui Fukuda; Ko Okumura; Hiroaki Miyajima; Yoshikazu Ohya
Original assignee: Koji Fukuda; Harui Fukuda; Ko Okumura; Hiroaki Miyajima; Yoshikazu Ohya
Priority date: 2002-06-28
Filing date: 2003-06-27
Publication date: 2004-01-08
Also published as: JP2004083555A; AU2003243011A1

Abstract

The invention provides for an agent for curing anaphylactic disease, which is obtainable by mixing shoots of plant belonging to the family Pinaceae with water and saccharide and then fermenting them. Further, the agent can be produced by using a fermentation product obtained by yeast isolated from the agent.

Description

DESCRIPTION

AGENT FOR CURING ANAPHLACTIC DISEASE AND HEALTH FOOD, AND MANUFACTURING PROCESS THEREOF

Technical Field

The present invention relates to an agent for curing anaphylactic disease such as an anaphylactic shock and food dependent exercise induced anaphylactic, and a manufacturing process of the agent. Further, the present invention relates to a health food for improving a symptom of anaphylactic disease, and a manufacturing process of the health food.

Background Art

A human body has an immune system that is a defense mechanism. And, when extraneous substances such as bacteria and viruses invade body, the system antagonizes them and protects body. Allergies are caused due to excess action of this immune system. Recently, increasing number of people are suffering from allergies possibly because of, though the details are not clear, air pollution, change of dietary life, physical or mental stress increase, environmental changes such as room pollution and the like due to change in resident circumstances, or change in human body constitution.

The allergic diseases include asthma, atopic dermatitis, allergic conjunctivitis, allergic rhinitis such as pollenosis, food allergy, anaphylactic shock caused by drugs and food dependent exercise induced anaphylactic.

The first remedy for these allergic diseases is to avoid the allergens, though the remedy is rather passive. When dusts or mites in a house are the allergens, the house must be cleaned to remove the allergens, and when a pollen is the allergen, in the season of the pollen floatation, going out should be held or a mask should be worn to prevent suction of the allergen, and in the case of a food allergy, the food causing the allergy should not be eaten. However, such a passive remedy is troublesome to the patient and significantly restricts the activities of the patient.

For remedy of asthma, the attack of asthma is stopped or is prevented by symptomatic treatments, and for these purposes, medicines such as sympathetic nervous drugs such as adrenalin, adrenal cortical steroid hormones, theophyllin drugs and the like are used. Though asthma is a disease sometimes leading the patient to death, there is still no remedy for complete recovery.

In infant period, atopic dermatitis causes distresses not only to the patient but also to the parents of the patient. Though the most of the patients are cured before maturity, there is a case where the dermatitis lasts to the adulthood. In such a case, adolescent men and women are distressed by thickening of the skin of face, breast, inside of elbow and knee and by severe itching. As the remedy for this, topical treatments are the major methods to treat the patient, and depending on the symptom, adrenal cortical hormones, antihistamine drugs and other anti-inflammatory agents are used. When itching is strong, systemic application of an antihistamine agent would be necessary.

Effective drugs for medical treatment often cause anaphylactic shock. Therefore, it often happens that the treatment for a patient having drug allergy is restricted. There are some cases where drugs such as antibiotics, insulin and contrast mediums are restricted for a patient. If the patient does not have enough information about those drugs, he may develop a serious anaphylactic shock by mis-taking those drugs, and it may be fatal to the patient.

In recent years, increasing interest in food-dependent exercise induced anaphylactic shock has been emerged. A patient with a food dependent exercise induced anaphylactic shock develops anaphylactic shock by exercising after taking specific foods. Many patients are diagnosed as teenage boys during puberty. Clinical symptoms are a fever, itch, red spot, a nettle rash, edema, stomachache, diarrhea and vomiting. As a serious case, the patient may die of breathing difficulties caused by edema of larynx, blood pressure dropped or losing consciousness.

Conventionally, a treatment for anaphylactic disease is to avoid drugs and foods that cause anaphylactic shock and to administer drugs such as adrenalin or steroid when anaphylactic shock occurs.

However, such treatments just suppress the symptoms, they cannot cure allergic diseases including anaphylactic disease completely, and the drugs may produce side effects.

When an allergen can be specified, there is a treatment called a sensitivity-reducing remedy in which the extract of the allergen is, first, injected to the patient hypodermically in very small amount, then, the amount is gradually increased to give a resistance against the allergen to the patient. However, in this remedy, the injection of the extract must be repeated on the patient periodically such as once to twice a week and the remedy requires a long period of time, additionally, an effect may not be sufficient enough in some patients, further, anaphylactic shock sometimes leading the patient to death may be caused in some incidents.

For remedy of asthma and atopic dermatitis, there are a lot of folk medicines, however, such medicines may worsen the symptom in some cases.

Disclosure of Invention

The present invention relates to an agent for curing anaphylactic disease without side effects by taking the agent for a short period, and a health food and manufacturing process thereof.

One aspect of the invention relates to an agent for curing anaphylactic disease which is obtainable by mixing shoots of plants belonging to the family Pinaceae with water and saccharides, and then fermenting the mixture. Another aspect of the invention relates to an agent that is effective in curing anaphylactic disease such as an anaphylactic disease caused by drugs and food dependent exercise induced anaphylactic disease. As the plants belonging to the family Pinaceae, plants belonging to the genus Pinus is preferable, and as the saccharides, sugar is preferable.

Further, another aspect of the invention relates to an agent for curing anaphylactic disease which is obtainable by mixing shoots of pine leaves with water and sugar, and then fermenting the mixture. Further one aspect of the invention relates to an agent that is effective in curing anaphylactic disease such as an anaphylactic disease caused by drugs or food dependent exercise induced anaphylactic disease.

Further, another aspect of the invention relates to a manufacturing process of a drug for curing anaphylactic disease, which comprises the steps of: (1) dissolving saccharides in sterilized water to obtain a saccharide solution and (2) adding shoots of plants belonging to the family Pinaceae to the solution and fermenting them. As the plants belonging to the family Pinaceae, plants belonging to the genus Pinus is preferable, and as the saccharides, sugar is preferable. Further, one aspect of the invention relates to a manufacturing process of a drug, for curing anaphylactic disease, which comprises the steps of: (1) dissolving sugar in sterilized water to obtain a sugar solution and (2) adding shoots of pine leaves to the solution and fermenting them.

The fermentation can be conducted under anaerobic conditions, preferably at 10-70°C, more preferably at 20-70°C, preferably for 3-9 months, more preferably for 4-8 months.

Further, one aspect of the invention relates to a manufacturing process, which comprises the steps of: (1) dissolving sugar in hot water and cooling them to a room temperature to prepare a sugar solution, and (2) adding washed shoots of pine leaves to the solution, sealing a container including them, and then fermenting them. The fermentation is preferably conducted in direct sunlight until the beginning of winter.

Further, one aspect of the invention relates to a health food (including a health beverage) for improving anaphylactic disease, which is obtainable by mixing shoots of plants belonging to the family Pinaceae with water and sugar, and then fermenting them. Further, one aspect of the invention relates to a use of a fermentation product manufactured by above process, that is, yeast isolated from the agent, for a drug, health food. One aspect of the invention provides an agent for curing anaphylactic disease without side effects by taking the agent for a short term, and a health dependent exercise induced by anaphylactic disease.

One aspect of the invention cures allergy type I disease represented by anaphylactic disease such as an anaphylactic shock and a food dependent exercise induced anaphylactic disease. Accordingly, one aspect of the invention provides an agent, health food and health beverage that are significantly effective in curing or improving anaphylactic disease.

The yeast was disclosed in international patent application (WO/01/95922) of the present applicant, and has been deposited as HARUISAN A-3 with Independent Administrative Agency, National Institute of Advanced Industrial Science and

Technology (AIST), International Patent Organism Depositary, Chuo No. 6, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture (old name: National Institute of Bioscience and Human-Technology National institute of Advanced Industrial Science and Technology, Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, name has been changed on April 1, 2001) on March 12, 2001, and specified by deposit No. FERM BP-7499. A series of yeasts having the microbiological properties equivalent to the deposited yeast are also included in the present invention. Further, in the present invention, a fermentation product obtained by the fermentation using the above-mentioned yeast is included, and as the fermentation product, pharmaceutical preparations such as allergic curative medicines, health foods, health drinks, and raw materials of cosmetics, and the like are exemplified. Here, the health food and health drink indicate foods and drinks such as supplements and the like used for the purpose of improving body constitution and maintaining health.

The present disclosure relates to subject matter contained in Japanese Patent Application No. 2002-190091, filed on, June 28, 2002 and No. 2003-49555, filed on February 26, 2003, the disclosure of which are expressly incorporated herein by reference in its entirety.

Brief Description of Drawings

Fig. 1 is a microscopic photograph (differential interference, X 2400) showing an example of the ascospore of isolated yeast isolated from the agent for curing anaphylactic disease (fermentation product) of the present invention. As the medium, malt extract agar medium was used.

Fig. 2 shows the results of the mutagenicity screening tests made on an agent for curing anaphylactic disease of the present invention, where base pair-substituted type strains are used (TA100: D, TA1535: 0, WP2uvrA: Δ). In the figure, "A" represents a result in the case of utilizing no metabolism activation (-S9), and "B" represents a result in the case of utilizing metabolism activation (+S9), respectively. Fig. 3 is a graph showing the results of the mutagenicity screening test of on an agent for curing anaphylactic disease of the present invention, and shows the results obtained by using a frame shift type strain (TA98: D, TA1537: O). In the figure, "A" represents a result in the case of utilizing no metabolism activation (-S9), and "B" represents a result in the case of utilizing metabolism activation (+S9), respectively. Fig. 4 is a photograph that shows an example of the PCA reaction in a rat that was administered an agent for curing anaphylactic disease of the invention. In the Figure, rats in group C are the control group and were orally administered distilled water which is injection graded distilled water, and rats in group D were orally administered an agent for curing anaphylactic disease. "Anti-DNP-IgE SPE-7 titer" refers to the final dilution ratio of antigen specific IgE antibody. Best Mode for Carrying Out the Invention

The agent for curing anaphylactic disease of the present invention is obtainable by mixing shoots of plant belonging to the family Pinaceae with water and saccharides and them fermenting them.

As the plant belonging to the family Pinaceae that can be used in the present invention, exemplified are Abies firma Sieb. & Zucc, Abies homolepis Sieb. & Zucc, Abies mariesii M.T. Mast., Abies sachalinensis (Friedr, Schmidt) M.T. Mast, var marie, Abies sachalinensis (Friedr, Schmidt) M.T. Mast., Abies veitchii Lindl., Cedrus deodara (Roxb. ex D. Don) G. Don, Larix gmelini (Rupr.) Kuzeneva, Larix Kaempferi (Lamb.) Carriere, Picea abies (L.) Karst., Picea glehnii (Friedr. Schmidt) M.T. Masters, Picea jezoensis (Sieb. & Zucc.) Carriere var. hondoensis, Picea jezoensis (Sieb. & Zucc.) Carriere, Picea koyamae Shirasawa, Picea polita (Sieb. & Zucc.) Carriere, Pinus x densi-thunbergii Uyeki, Pinus densiflora Sieb. & Zucc, Pinus densiflora Sieb. & Zucc. cv. Umbraculifera, Pinus koraiensis Sieb. & Zucc, Pinus palustris Mill., Pinus parviflora Sieb. & Zucc. var. parviflora, Pinus parviflora Sieb. & Zucc. var. pentaphylla (Mayr) Henry, Pinus pumila (Pall.) Regel, Pinus rigida Mill., Pinus strobus L, Pinus sylvestris L., Pinus teada L, Pinus thunbergii Park, Pseudotsuga japonica (Shiras.) Beissn., Tsuga diversifolia (Maxim.) M.T. Mast., Tsuga Sieboldii Carriere and the like. Of them, Pinus x densi-thunbergii Uyeki, Pinus densiflora Sieb. & Zucc, Pinus densiflora Sieb. & Zucc. cv. Umbraculifera, Pinus koraiensis Sieb. & Zucc, Pinus palustris Mill., Pinus parviflora Sieb. & Zucc. var. parviflora, Pinus parviflora Sieb. & Zucc. var. pentaphylla (Mayr) Henry, Pinus pumila (Pall.) Regel, Pinus rigida Mill., Pinus strobus L., Pinus sylvestris L, Pinus teada L. and Pinus thunbergii Parl. which are plants belonging to the genus Pinus are preferable, and particularly, Pinus densiflora

Sieb. & Zucc, Pinus densiflora Sieb. & Zucc. cv. Umbraculifera, Pinus koraiensis Sieb. & Zucc, Pinus palustris Mill., Pinus pumila (Pall.) Regel, Pinus thunbergii Parl. and the like are generally grown pine trees, and also preferable from the standpoint of easy availability.

As the saccharides can be used in the present invention, sucrose, invert sugar, maltose and the like are exemplified. Among them, sucrose is preferable from the standpoint of easy availability, and as the sucrose used, any sugar such as white sugar, black sugar, yellow soft sugar, beet sugar, millet sugar and the like can be used, and white sugar is preferable.

As the water can be used, previously sterilized water is preferably used to prevent proliferation of saprophytic bacteria, and as the sterilization method, any known methods generally employed to sterilize water can be used, for example, water can be sterilized by boiling and the like.

The agent for curing anaphylactic disease of the present invention can be obtained by dissolving saccharides in the above-mentioned sterilized water to prepare a saccharides solution, adding to the saccharides solution a shoots of plant belonging to the family Pinaceae, and fermenting the mixture. The shoots of plant belonging to the family Pinaceae to be added to said aqueous solution may be a shoots collected from any kind of plants belonging to the family Pinaceae, and particularly, a shoots collected from a plant belonging to the genus Pinus is preferable. The preferable season for collecting the shoots is the season after completion of blooming of the plant, and the shoots collected in this season has the highest effectiveness as an agent for curing anaphylactic disease and therefore is preferable. In the case of pine tree, though it depends on climate of the land, reddish female flower blooms at the peak of a branch and yellow male flower blooms around the new branch generally around early April to late June, therefore, the shoots at the peak of the branch are preferably collected and used when blooming of these flowers is completed.

For preparing the starting solution used in the fermentation, about 0.5 kg of saccharide is dissolved in 1 liter of water. Then, to the resulting solution, about 25 shoots of plant belonging to the family Pinaceae are added. In this case, the saccharide is not required to be completely dissolved and there is no problem if the saccharide presents remaining undissolved in the solution. The starting solution to be fermented may advantageously contain shoots of plants belonging to the family Pinaceae such as shoots of pine leaves in the ratio set forth in the above, and there is no problem if it contains leaves and flowers other than the shoots.

The fermentation can be conducted under anaerobic conditions and the fermentation is effected by allowing the starting solution to stand still at 10 to 70°C, preferably 20 to 60° C for 3 to 9 months, preferably 4 to 8 months. As the anaerobic condition, for example, the starting solution is filled into a light-shielded vessel and the like and the vessel is sealed air tightly. The fermentation is realized by placing this sealed vessel at a place receiving direct sunlight until around early winter. After the period of spontaneous fermentation is completed, the vessel is opened, and the solid materials such as shoots of plant belonging to the family Pinaceae are removed to obtain an agent for curing anaphylactic disease of the present invention. The above-mentioned early winter is determined in consideration of the shoots collecting period, namely, charging period, and if the blooming period is from early April to early May and if the shoots collection period after completion of blooming is from middle May to early June in the producing area, the period of fermentation completion shall be early winter (middle November), however, this is only one example, and this period can optionally be changed.

The agent for curing anaphylactic disease of the present invention is effective in widely suppressing the allergic reaction related to anaphylactic disease and allergic disease that is caused by a chemical mediator when the mediator is released from a cell including a mast cell and basophilic leukocyte, as shown below. Therefore, the agent is effective in curing anaphylactic disease but also allergic disease. In view of the mechanism of action, the agent is believed to be effective in curing type I allergic disease such as asthma, atopic dermatitis, allergic rhinitis, acute urticaria, pollenosis, food allergy in addition to the above-mentioned diseases

The agent for curing anaphylactic disease of the present invention uses a fermentation product per se obtained by the spontaneous fermentation, however, a sweetener and flavoring agent may be added to the product to improve the acceptance of the product in drinking, or various additives such as a preservative and the like may be added for storing the product for a long period of time or for other purposes.

For the application of the agent for allergies of the present invention, in the case of adult, in general, about 30 to 50 ml of the agent is administered twice or three times a day. In the case of child, half dosage of that in the adult administration may be used. Since the agent for allergies of the present invention has no toxicity and no mutagenicity and therefore is safe, there is no problem if an amount over the above-mentioned dosage is used.

The fermentation product of the present invention obtained by the fermentation can be used not only as an agent for allergies but also as health food or health beverage. The health food or health beverage are not used mainly for treatment, but used to improve body constitution and maintain healthy condition. Accordingly, a health food and a health beverage for improving anaphylactic disease of the present invention improve anaphylactic disease, that is, they can be used for alleviating or preventing from the disease. In this case, it is preferable, in view of inclination of consumers, to add the above-mentioned sweetener, flavoring agents or the like to make the product acceptable to drink or eat and to make the product compatible with the inclination.

Further, the present invention provides an agent for curing anaphylactic disease, a health food for improving anaphylactic disease and a manufacturing process of them, which contain a product using yeast that is isolated from the fermentation product or an extract of the product.

Isolation of the yeast is also described in WO 01/95922. Specifically, a fermentation product that is the agent for curing anaphylactic disease of the present invention was used as the specimen. GPLP agar plate culture method was used to culture the microorganisms on the plate, and the colonies dominantly grown on the culture plate was picked up to obtain the isolated yeast. On the isolated yeast, the morphological and physiological observations were performed, and the yeast was identified by consulting literatures (Kurtzm an, C. P. et at., "The yeasts, A Taxonomic Study" 4-th edition (1998), Elsevier Science B. V. ; Baraett, J. A. et al., "Yeasts: Characteristics and identification" , 3-rd edition (2000), Cambridge University Press, these literatures are incorporated as parts of the specification of the instant application by reference). The total number of yeast in the fermentation product was 1.4xl0⁵/g. The results form the morphological and physiological observations on the isolated yeast are shown in Table 1 and an example of ascospore of the isolated yeast is shown in Fig. 1.

Table 1: Properties of the Isolated Yeast

Item Observed Result

Form of nursing cell Oval to ellipse Proliferation form Multiple budding Liquid culture Sedimentation observed, no film formation observed (25°C, 3 days)

Pseudohypha Formed (25°C, 3 days) Ascospore Mating observed between individual nursing cells, 1 to 4 ascospores in the form of ellipse formed, ascus not divided (see

Fig. 1)

Utility of nitrogen source: Nitrate Ethylamine Growth in vitamin-deficient medium Growth in the presence of NaCl: 10% + 12.5% + 15% Weak 16% Growth in the presence of cycloheximide: 0.1% 0.01% Growth in the presence of 1% acetic acid Growth at 37°C Degradation of urea Coloration of DBB Fermentation property:

Glucose

Galactose

Sucrose

Maltose

Lactose

Raffinose

Trehalose

Utilization of carbon source:

Glucose

Galactose

Sucrose

Maltose Cellobiose Trehalose Lactose Melibiose Raffinose Melezitose

Starch

D-xylose

L-arabinose

D-ribose

L-rhamnose

D-glucosamine

N-acetyl-D-glucosamine

Ethanol +

Glycerol +

Erythritol

Ribitol +

D-mannitol +

Citrate Inositol

According to the above results, the isolated yeast is identified as Zygosaccharomyces bisporus from the morphological and physiological properties. The Zygosaccharomyces bisporus belongs to ascomycetous yeast, mates between the individual cells and forms 1 to 4 ascospores of sphere to ellipse form. Further, Zygosaccharomyces bisporus is osmosis resistant yeast and isolated from fermentation foods, soft drinks and the like. Moreover, a DNA homology test was conducted between the isolated yeast and the type strain to give the following results. Namely, Zygosaccharomyces bisporus IFO 1131 and Zygosaccharomyces bailii LFO 1098 were used as the type strains according to Takayuki Ezaki et al., Japanese Journal of Bacteriology, vol. 45, p. 851 (1990) and Masaaki Takahashi et al., Tokyo University of Agriculture Isotope Center Study Report, No. 7, p. 69 (1993), both of which literatures are incorporated herein by reference. The DNA homology test between them and the isolated yeast was carried out by a photo biotin labeling method using a microplate, in a DNA-DNA hybridization test. The preparation of DNA was conducted according to Jahnke, K.-D. et al., Trans. Br. Mycol. Soc, Vol. 87, pp. 175-191 (1986)(this literature is incorporated as parts of the specification of the instant application by reference). The results are shown in Table 2.

Table 2: Results of DNA-DNA Hybridization Homology Test between Isolated Yeast and Type Strain

Type strain Homology (%)

Zygosaccharomyces bisporus IFO 1131 72

Zygosaccharomyces bailii IFO 1098 98

According to the results of the homology test with the above-mentioned type strains, though the yeast of the present invention is identified as Zygosaccharomyces bisporus from the morphological and physiological properties, DNA sequence itself is close to Zygosaccharomyces bailii rather than Zygosaccharomyces bisporus. Accordingly, it is recognized that the yeast of the present invention is a novel strain differing from both type strains.

The applicant named this novel yeast as "HARUISAN A-3" which is deduced to belong to Zygosaccharomyces genus. Presently, it is not clear whether this novel yeast "HARUISAN A-3" is just a novel strain or is a novel species or genus. However, the applicant deposited the novel yeast isolated and named "HARUISAN A-3" with Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology (AIST), International Patent Organism Depositary, Chuo No. 6, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture, Japan (old name: National Institute of Bioscience and Human-Technology National institute of Advanced Industrial Science and Technology, Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan, in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, on March 12, 2001, under a deposit number of FERM BP-7499. The above-mentioned Depositary was turned into an independent administration corporation on April 1, 2001, and altered as Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology (AIST). Thus, the name was changed on April 1, 2001.

The isolated novel yeast of the present invention is believed to significantly contribute to ef ectiveness of the agent for curing anaphylactic disease together with shoots of plants belonging to the family Pinaceae, and therefore is extremely useful yeast.

Next, the agent for curing anaphylactic disease of the present invention is described in detail by examples, but the scope of the invention is not limited to the following examples.

(Example 1: Production Example of Agent for Curing Anaphylactic Disease)

The agent for curing anaphylactic disease of the present invention can be produced, for example, by the following method.

Shoots of pine leaves of Pinus thunbergii Park, Pinus densiflora Sieb. & Zucc. and Larix kaempferi (Lamb.) Carriere were collected when pine blooming was completed (in Fukushima prefecture in Japan, around middle May to early June), and the collected shoots of pine leaves were washed thoroughly with water. White sugar was added and dissolved into hot water, and cooled to around room temperature, and the resulting sugar water and water-washed shoots of pine leaves were placed in a vessel, for example, a plastic vessel, the vessel is sealed, and the vessel is placed at a place receiving direct sunlight until early winter (around middle November, in Fukushima pref .) to cause spontaneous fermentation for the production. The vessel was opened around early winter, and the pine leaves were removed to obtain the agent for curing anaphylactic disease of the present invention.

For the sugar water used, about 1 kg of white sugar was used per about 2 liters of water, and 50 pine leaves were used per about 2 liters of water.

As described above, the production was conducted using 1 kg of white sugar and about 50 pine leaf shoots were used per about 2 liter of water, which resulted in about 1.2 liter (about 60% of the charged sugar water) of an agent for curing anaphylactic disease in the form of liquid having white turbidity. The components of the agent and the content were analyzed, and the results are shown in Table 3.

Table 3: Analyzed Values of Components of Agent for Anaphylactic Disease

Component Content Analysis method used

Moisture 98.0% Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin", normal pressure heating method

Protein 0.0% Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin", Nitrogen quantification conversion method

Lipid 0.4% Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin", ether extraction method (Soxhlet method) Ash 0.1% Guidelines for food hygiene inspection

"Shyokuhin Eisei Kensa Shishin", direct ashing method

Saccharides 1.5% Guidelines for food hygiene inspection

"Shyokuhin Eisei Kensa Shishin", Calculated.

Crude fiber 0.0% Guidelines for food hygiene inspection

"Shyokuhin Eisei Kensa Shishin", modified

Henneberg Stoman method

Dietary fiber 2.4% Guidelines for food hygiene inspection

"Shyokuhin Eisei Kensa Shishin", Prosky method

Na 5.1 mg/100 g Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin"

Less than 0.50 Guidelines for food hygiene inspection mg/100 g*¹ "Shyokuhin Eisei Kensa Shishin"

Fe 0.11 mg/100 g Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin"

Ca 1.66 mg/100 g Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin"

K 18.3 mg/100 g Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin"

General bacteria 2700 c.f.u./g Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin"

Escherichia coli Negative Guidelines for food hygiene inspection "Shyokuhin Eisei Kensa Shishin"

Thiamine Less than 0.01 HPLC method mg/100 g*¹

Riboflavin Less than 0.01 HPLC method mg/100 g*¹

Ergosterol 0.07 mg 100 g HPLC method

Niacin 0.04 mg/100 g Bioassay using microorganism*² β-glucan 0.03 mg/100 g Enzyme method

*1 less than detection limit

*2 Strain used: Lactobacillus plantarum ACTT 8014

This analysis was conducted only on the above-mentioned inspection items mainly regarding general food, and there is a possibility that the agent for curing anaphylactic disease of the present invention contains other components than the above components. Therefore, it could not be specified which component was particularly important as an agent for curing anaphylactic disease, however, the agent for curing anaphylactic disease was effective in treating allergies. Next, with respect to a plurality of remedies for allergies of the present invention produced as described above, number of fungi, number of yeast and number of general bacteria (viable bacteria) contained in the agent for curing anaphylactic disease were counted. The number of fungi and the number of yeast were measured by using GPLP agar plate culture method, and the number of general bacteria (viable bacteria) was measured by using the anti-fungus agent-added SCDLP agar plate culture method in two ways, namely, pH of the medium was controlled to 3.5 using tartaric acid; pH of the medium was not controlled. The results are shown in Table 4. Table 4: Number of Microorganisms Contained in the Agent for Anaphylactic Disease Microorganism Fermentation Fermentation Fermentation Fermentation product 1 product 2 product 3 product 4

Number of Number/0.1 Negative Negative Negative Negative fungi g

Number of Number/ g 1.3xl0² 3.3xl0⁵ 1.6x10^ 3.3xl0⁴ yeast

Number of Number/ g 100 or less 100 or less general bacteria

Number of Number/ g 100 or less 100 or less 100 or less 100 or less general bacteria, pH controlled

According to the above results, the growth of fungus was not recognized in the agent for anaphylactic disease of the present invention, and the number of general bacteria (viable bacteria number) was also extremely small. Further, it can be understood that yeast was present in the order of 10² to 10⁵/g in the fermentation product, though the yeast concentration was slightly irregular depending on the lot of the fermentation product. The yeast existed in the above fermentation products corresponded to the deposited novel yeast.

(Test Example 1: Oral Toxicity of Agent for Curing Anaphylactic Disease)

Next, the oral toxicity of the agent for anaphylactic disease of the present invention was examined. The stock solution of the agent for curing anaphylactic disease of the present invention and that obtained by adding honey (10 wt%) to the stock solution were used as the specimens. Two test groups each consisting of 5 male and 5 female SD [Crj: CD(SD)IGS] rats were administered the stock solution or the honey added stock solution at a rate of 2000 mg kg and the control group was administered the injection water (dose: 0 mg kg) alone. The specimens and the injection water were forcibly administered to the rats orally by using a disposable syringe (volume: ImL) equipped with a per os stomach conductor. The toxic symptom and approximate lethal dose were investigated over the period of 15 days after the administration (including administration day).

Throughout the test period, no death incident was observed in either of male and female rats in the group orally administered the agent for curing anaphylactic disease of the present invention and in the group administered the specimen prepared by adding honey to the agent at the rate of 2000 mg/kg, including the control group. Moreover, no change ascribed to administration of the specimens was recognized in general condition, body weight and autopsy of the rats. From the above results, it was concluded that the approximate lethal dose of the agent for curing anaphylactic disease of the present invention under the conditions of this test was 2000 mg/kg or more for both male and female.

(Test Example 2: Mutagenicity of Agent for Curing Anaphylactic Disease) Next, the mutagenicity of the agent for curing anaphylactic disease of the present invention was investigated. Regarding the mutagenicity of the agent for curing anaphylactic disease of the present invention, the agent was applied to the histidine-dependent Salmonella typhimurium, TA98, TA100, TA1535 and TA1537 strains, and tryptophan-dependent Escherichia coli,WP2uvrA strain according to the revised plate method of Ames et al. (Maron, D.M. et al., "Revised methods for the Salmonella mutagenicity test" , Mutation Res., Vol. 113, pp. 173 to 215 (1983), this literature is incorporated as parts of the specification of the instant application by reference), and the mutagenicity was investigated under the presence of the metabolism activation or the absence of the metabolism activation.

Those tests were conducted at a dose of 312.5, 625, 1250, 2500 and 5000 μg/plate of the agent of the present invention. As the results, the number of the reverse mutation colonies of each test strain in the specimen group showed no increase in the dose-dependent manner and no increase over 2-fold or more was observed as compared to the negative control, irrespective of presence or absence of metabolism activation system. Further, no growth inhibition and no precipitation of the specimen were recognized. The results are shown in Figs. 2 and 3. Fig. 2 shows the result in the base pair-substituted type strain (TA100: D, TA1535: 0, WP2uvrA: Δ). In the figure, "A" represents the result in the absence of the metabolism activation, and "B" represents the result in the presence of the metabolism activation to which S9 mix was added. Fig. 3 shows the results in the frame shift type strain (TA98: D, TA1537: O). In the figure, "A" represents the result in the absence of the metabolism activation, and "B" represents the result in the presence of the metabolism activation to which S9 mix was added. As the negative control substance, distilled injection water that was used as the solvent to prepare the specimen was administered. As the positive control substance, compounds: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Aminoanthracene (2-AA),Sodium azide (SA) and 9-Aminoacridine (9-AA), were used. AF-2, 9AA and 2-AA were dissolved in DMSO and SA was dissolved in distilled injection water, respectively, and used depending on the strain and on the presence or absence of the metabolism activation.

From the above results, it was judged that the mutagenicity of the agent for curing anaphylactic disease of the present invention under the conditions of this test was negative.

(Test Example 3: Medicinal Action of Agent for Curing Anaphylactic Disease) The rats were given an epidermal of anti-DNP-IgE under the skin for intracutaneous sensitization, the agent for curing anaphylactic disease of the invention was administered orally, and then an antigen (DNP-BSA) and Evans blue were administered intravenously to a coccygeal vein, to confirm the medical action of the agent by the PCA reaction.

That is, six Wister male rats of 12 weeks old obtained from Nippon Charles River Co., Ltd. were given an epidermal of 100 H 1 of monoclonal anti dinitrophenyl-IgE antibody (anti-DNP-IgE antibody) clone: SPE-7 (produced by Sigma Co., Ltd.; Catalog No.D-8406, Lot No.l00K-4850) diluted solution into the back for intracutaneous sensitization. After the sensitization, the agent for curing anaphylactic disease of the invention of Example 1 was administered orally for four days at the rate of 1 ml/day.

Only sterilized water of injection grade was administered orally to the control group (See Table 5).

Table 5 Test Groups

Number of Number of Days

Test Group Tested Substance Tested Animals of Administration

C (Controls) Distilled Water for Injection 3 4 D (Subjects) Curing Agent By Example 1 3 4

After administering the test substances, dinitrophenyl-binding bovine albumin (DNP-BSA) and Evans blue, which are the PCA reaction inducing substances, were administered intravenously to a coccygeal vein.

The results were obtained by using the end point method, that is, the method based on the results of the PCA reaction at the final degree of dilution of anti-DNP-IgE. The symbol (+) denotes that the reaction was seen clearly inside the skin, (±) denotes that the reaction was seen unclearly, and (-) denotes that the reaction was not seen. The results are shown in Table 6. Fig. 4 shows examples of the reaction inside the skin.

Table 6 Results of PCA Reactions

Final Dilution Ratio of anti-DNP-IgE Group X2000 x4000 x8000 xlόOOO x32000 x64000

C-l + +

C-2 + +

C-3 + +

D-l + +

D-2 + -

D-3 + _

Further, after the sensitization of anti-DNP-IgE under the skin, the leaked pigment (Evans blue) was extracted from spots under the skin. According to the amount of the pigment the degree of local reaction was determined. Table 7 shows the results.

Table 7 Amount of Pigment Indicating Degree of PCA Reaction

Final Dilution Ratio of anti-DNP-IgE

Group x2000 x4000

C-l 1.67 0.65 C-2 2.93 0.79 D-l 0.69 0.33 D-2 0.54 0.21

The final dilution ratio in the PCA reaction was x4000 in the control group C, and x2000 in group D which was orally administered the agent for curing anaphylactic disease of the invention, the PCA reaction was seen to be suppressed significantly.

Also, the dilution ratio of the monoclonal anti dinitrophenyl-IgE antibody is x2000, x4000, x8000, xlόOOO, x32000 and x64000 corresponding to 500 ng/100 Ail, 250 ng/100 μ.1, 125 ng/100 1, 63 ng/100 At 1, 31 ng/100 μ.1 and 16 ng/100 β 1.

At least the results show that the agent for curing anaphylactic disease of the invention suppresses the reaction related to anaphylactic or allergic reaction after a chemical mediator is released from a cell such as a mast cell and basophilic leukocyte.

(Test Example 4: Confirmatory Study of Amount of Agent for Curing Anaphylactic Disease)

To examine an effective amount of the agent, the PCA reaction was performed in the same manner as in Test Example 3.

That is, each rats was given an epidermal of 100 At 1 of monoclonal anti DNP-Ig antibody (SPE-7) diluted solution into six points on the back for intracutaneous sensitizatiion, and the agent for curing anaphylactic disease of the invention was administered orally for four days at the rate of O.lml/day, 0.5 ml/day and 1.0 ml/day, respectively.

Sterilized water of injection grade was administered orally for four days at the rate of 1.0 ml/day to the control group.

The next day of the final administration, dinitrophenyl-binding bovine albumin (DNP-BSA) and Evans blue as the PCA reaction-inducing substances were administered intravenously to a coccygeal vein, and the leakage of pigment "Evans blue" around the IgE sensitized area was determined by using the end point method 30 minutes after the administration.

Table 8 Test Groups Number of Number of

Test Group Tested Substance

Tested Animals Administrations

Distilled Water for

E (Controls)

Injection (1.0 ml)

Curing Agent

F (Subjects)

By Example 1 (0.1 ml)

Curing Agent

G (Subjects)

By Example 1 (0.5 ml)

Curing Agent

H (Subjects)

By Example 1 (1.0 ml)

Table 9 Results of PCA Reactions

Final Dilution Ratio of anti-DNP-IgE

Group X2000 X4000 X8000 X16000 X32000 X64000

E-l + + + -

E-2 + + + +

F-l F-2 F-3

G-l + G-2 + G-3

H-l H-2 + H-3 +

According to Table 9, the control group (group E) shows the PCA reaction up to x8000-16000 of dilution (63-125ng). In contrast, the PCA reaction was seen up to X4000-8000 of dilution(125-250ng) in group F at the rate of 0.1 ml/day, the PCA reaction of IgE was shown up to x2000-4000 of dilution (250-500ng) in group G at the rate of 0.5ml/day and in group H at the rate of l.Oml/day. Also, concerning to the comparison between the control group E and group F, the suppression of the PCA reaction in group F was calmer, but the effect of the suppression was seen stronger. Further, groups G and H have similar results, however, according to the observation of the leakage of the pigment, the amount of the pigment leaked was less in group H than in group G. The weight of rats used in the test was about 250g, and considering this, the dosage for a human (having a weight of 60kg) is calculated to be 24 ml/day, 120 ml/day and 240 ml/day. However, it is preferable to administer more than 120 ml/day to ensure the effect of the treatment.

(Test Example 5: Fermentation Period and Medical Action of Agent for Curing Anaphylactic Disease)

Next, the effects that the fermentation period exerts medical action were examined.

That is, by using the drugs fermented for 1,3 and 5 months, medical effects were determined by the PCA reaction similarly to Example 3 as follows. Rats were given an epidermal of 100 Ai l of a diluted solution of mouse monoclonal anti DNP-IgE antibody (SPE-7) into six points on the back for intracutaneous sensitization Then, the agents for curing anaphylactic disease of varying fermentation terms were administered orally at the rate of l.Oml/day for four days. Sterilized water for injection was administered orally at the rate of 1.0 ml/day for four days to the control group.

On the next day of the final administration, dinitrophenyl-binding bovine albumin (DNP-BSA) and Evans blue as the PCA reaction inducing substances were administered intravenously to a coccygeal vein, and the leakage of pigment Evans blue around the IgE sensitized area was determined by using the end point method 30 minutes after the administration. Details of tested groups and results are shown in Tables 10 and 11.

Table 10 Test Groups

Number of Number of

Test Group Tested Substance

Tested Animals Administrations

Distilled Water

I (Controls) for Injection (1.0 ml) Curing Agent Fermented

J (Subjects) for 1 Month (1.0 ml) Curing Agent Fermented

K (Subjects) for 3 Months (1.0 ml) Curing Agent Fermented

L (Subjects) for 6 Months (1.0 ml)

Table 11 Results of PCA Reactions

Final Dilution Ratio of anti-DNP-IgE

Group x2000 x4000 x8000 xl6000 x32000 x64000

1-1 + +

1-2 + +

J-l + +

J-2 + +

J-3 + +

K-l K-2 + +

K-3 + +

L-l - -

1_^2 + +

L-3 + _m

According to Table 11, the control (group I) showed the PCA reaction up to x8000 (125ng). In contrast, group J, fermented for one month, suppresses up to x8000 of IgE which is almost the same as the control group. Therefore, the suppression effect of the PCA reaction is rarely perceived in group J. However, groups fermented for three or six months are perceived to show a suppression effect on the PCA reaction, it is understood that the fermentation period is preferably three months, more preferably six months.

Claims

1. An agent for curing anaphylactic disease, which is obtainable by mixing shoots of plant belonging to the family Pinaceae with water and saccharide and then fermenting them.

2. The agent for curing anaphylactic disease according to Claim 1 wherein the plant belonging to the family Pinaceae is a plant belonging to genus Pinus.

3. The agent for curing anaphylactic disease according to Claim 1 wherein the saccharide is sugar.

4. An agent for curing anaphylactic disease, which is obtainable by mixing shoots of pine leaves with water and sugar and them fermenting them.

5. A manufacturing process . of a drug for curing anaphylactic disease, which comprises (1) dissolving saccharides in sterilized water to obtain a saccharide solution and (2) adding shoots of plants belonging to the family Pinaceae to the solution and fermenting them.

6. The manufacturing process according to Claim 5, wherein the plant belonging to the family Pinaceae is a plant belonging to genus Pinus.

7. The manufacturing process according to Claim 5, wherein the saccharide is sugar.

8. The manufacturing process according to Claim 5 wherein the fermentation is conducted under anaerobic conditions at 10 to 70°C for 3 to 9 months.

9. A manufacturing process of a drug for curing anaphylactic disease which comprises the steps of: (1) dissolving sugar in sterilized water to obtain a sugar containing solution and (2) adding shoots of pine leaves and fermenting them.

10. The manufacturing process according to Claim 9 wherein the fermentation is conducted under anaerobic conditions at 10 to 70°C for 3 to 9 months.

11. A manufacturing process of a drug for curing anaphylactic disease, which comprise the steps of: (1) dissolving sugar in hot water and cooling them to a room temperature to prepare a sugar containing solution, and (2) adding washed shoots of pine leaves to the solution, sealing a container including them, and then fermenting them.

12. The manufacturing process according to Claim 11 wherein the fermentation is conducted under anaerobic conditions at 10 to 70°C for 3 to 9 months.

13. The manufacturing process according to Claim 9 wherein the fermentation is conducted in direct sunlight.

14. A health food for improving anaphylactic disease, which is obtainable by mixing shoots of plant belonging to the family Pinaceae with water and saccharide and then fermenting them.

15. A health food for improving anaphylactic disease, which is obtainable by mixing shoots of pine leaves with water and sugar and them fermenting them.

16. A manufacturing process of an agent for curing anaphylactic disease, which uses yeast deposited under deposit number FERM BP-7499, or yeast having the equivalent microbiological property to that of the yeast deposited under deposit number FERM BP-7499.

17. A manufacturing process of a health food for improving anaphylactic disease, which uses yeast deposited under deposit number FERM BP-7499, or yeast having the equivalent microbiological property to that of the yeast deposited under deposit number FERM BP-7499.

18. A manufacturing process of a health beverage for improving anaphylactic disease, which uses yeast deposited under deposit number FERM BP-7499, or yeast having the equivalent microbiological property to that of the yeast deposited under deposit number FERM BP-7499.