WO2004067019A1 - Antiallergic agent containing ground lotus or lotus extract - Google Patents

Abstract

It is intended to provide an antiallergic agent, an antiallergic food additive and an antiallergic food each containing ground lotus or a lotus extract. Because of containing ground lotus, a lotus extract or a combination thereof as the active ingredient, these antiallergic agent, antiallergic food additive and antiallergic food make it possible to relieve and treat allergic diseases typified by pollenosis, bronchial asthma and atopic dermatitis.

Description

 Description Antiallergic agent containing crushed or extracted lotus

 TECHNICAL FIELD The present invention relates to an antiallergic agent, an antiallergic food additive and an antiallergic food, comprising a crushed or extracted lotus (Ne 1 bile bonuci ferra) or an extract thereof. Background art

 Today, in many developed countries, diseases due to type I allergic reactions, such as hay fever, bronchial asthma or atopic dermatitis, are increasing, and are becoming a social problem.

 Among the five types of antibodies that humans have, the antibody called IgE is deeply involved in the type I allergic reaction, and allergic individuals produce this antibody more than normal non-allergic individuals. Tend. IgE production is promoted by interleukin 4 (IL-4) and interleukin 13 (IL-13) derived from type 2 helper T lymphocytes (Th2), It has been reported that this is suppressed by interleukin 2 (IL-2) and interferon-α (IFN-r) derived from T-lymphocytes (Th1). Therefore, in order to suppress the production of IgE and improve the allergic constitution, it is important to suppress the production of IL-4 IL-13 or to promote the production of IL-2 and IFN-r. In addition, since TNF (tumor necrosis factor) is also involved in the type I allergic reaction, suppressing the production of TNF is also effective in suppressing the inflammatory response.

Antihistamines, antiallergic agents, steroids and the like have been developed and used as pharmaceuticals in order to improve the above-mentioned diseases caused by type I allergic reaction. These drugs may worsen symptoms after long-term administration (rebound phenomenon) and act on the central nervous system It may be accompanied by side effects such as drowsiness caused by the drug and effects on the endocrine system due to transdermal absorption.

 On the other hand, for the purpose of safely improving the above-mentioned diseases without side effects, foods that have an anti-allergic effect among conventionally eaten foods have been searched (JP-A-61-291524, JP-A-Heisei 1-291524). 121217, JP-A-7-215884, etc.). Also already mentioned and widely recognized are hot tea extract extracted from tea leaves of sugar beet (Rub ussu av iss imu s) and hot water extracted from leaves of perilla (_erillafrutesc en s). Extracted perilla leaf extract.

 However, there is a problem that compounds used as pharmaceuticals often have side effects as described above. There are also food-derived anti-allergic materials such as bean tea extract and perilla leaf extract, but from the viewpoints of a different mechanism of action, more effective medicinal properties, and Z or economic advantages, other foods may be used. There is a need for anti-allergic materials derived from the Japanese. Disclosure of the invention

 The present inventors have found that a crushed lotus root extract or extract has an anti-allergic effect, and based on this, have completed the present invention.

 The present invention provides a bovine allergic agent, an antiallergic food additive and an antiallergic food, comprising a lotus crush or extract, or a combination thereof. The present invention also provides a hay fever improving agent, a hay fever improving food additive and a hay fever improving food, comprising a lotus crushed or extracted product, or a combination thereof.

Further, the present invention provides a serum IgE concentration-lowering agent, a serum IgE concentration-lowering food additive, and a serum IgE-lowering food, comprising a lotus crush or extract, or a combination thereof. provide. The present invention also provides an IL-14 production inhibitor, a food additive for suppressing IL-14 production, and a food for suppressing IL_4 production, comprising a crushed or extract of lotus, or a combination thereof.

 Furthermore, the present invention provides a TNF production inhibitor, a TNF production suppression food additive, and a TNF production suppression food comprising a lotus crushed or extracted product or a combination thereof.

 In the present invention, the term "Lotus" refers to Neelmbboneuciera, which belongs to the family Nymphae (Nymphaeaecae) and the subfamily Lotus (Ne1umboideae). The rhizomes of the lotus are generally called "lotus root" and are commercially available. In addition, as the lotus that is used as a crushed product or an extract in the present invention, any part of a lotus plant body can be used. For example, lotus rhizome, stalk, leaf, root, fruit, etc. These combinations include, but are not limited to. Preferably, the lotus used as the crushed material or extract is a lotus underground stem, stem, leaf or root, or a combination thereof, and more preferably a lotus rhizome, stem or leaf, or a combination thereof. And even more preferably the lotus rhizome.

 The lotus crushed material can be prepared by crushing the lotus by any method using a crusher, a mixer, a crusher or any other device. The crushing treatment may be performed by adding a solvent such as water, or may be a mode in which only the lotus is crushed. The crushed lotus material in the present invention may be one obtained by crushing lotus, or may be heated and / or dewatered before crushing, simultaneously with crushing, or after crushing. May be used. It does not matter whether the heating Z or dehydration treatment is performed.

Preferably, the lotus crushed product is obtained by crushing the lotus and then heat-treating it, and then drying it.It is also obtained by drying the lotus and then crushing it. After the crushing process, the lotus is heated and And drying treatment (the order of heating and drying does not matter), followed by crushing treatment, but is not limited thereto.

 The crushed material can be in any shape such as paste, solid, granular material, powder, liquid (including any state such as solution and suspension), and the crushed material having these shapes is It can be manufactured using any known method. For example, the crushed material may be directly manufactured from a lotus so as to have these shapes, or as described above, the dried crushed material obtained by drying once may be formed into these shapes. It is also possible to manufacture so that.

 When a dry treatment is performed in the preparation of the crushed lotus, any known method can be used as a dry treatment method, for example, a freeze-dry method and a far-infrared drying method. Examples thereof include a heat drying method including a near-infrared drying method, but are not particularly limited.

In the present invention, the lotus extract is not limited to an extract obtained by a so-called solvent extraction process in which components in the lotus are transferred to a solvent to obtain an extract. The extract obtained by directly removing any component of the lotus, such as the juice obtained by the method, without using a solvent or the like is also an extract according to the present invention. Further, the extract may be prepared at room temperature or under heating. For example, as an example of a lotus extract, a lotus juice obtained by squeezing a chopped or crushed lotus lotion, a lotus juice obtained by squeezing a chopped or crushed lotus lotion, Extracts obtained by solvent extraction of the shredded or crushed material with or without heating may be mentioned. Solvents that can be used for solvent extraction include one or more of water, ethanol, propylene glycol, n-butanol, ethyl acetate, chloroform and the like, alone or in combination of two or more. Preferably, the solvent used for the extraction is water. The extract can be concentrated or concentrated to dryness as necessary. The extract can be in any form, such as a paste, solid, granule, powder, liquid (including any state such as solution, suspension, etc.). Extracts having these shapes can be produced using any known method. When a dry process is performed in the preparation of the lotus extract, any known dry process can be used as the dry process, such as the freeze-dry method and the far-infrared drying method. Examples thereof include a heat drying method including a near-infrared drying method, but are not particularly limited.

 In the medicine, the food additive and the food according to the present invention, the crushed lotus extract and the extract may each be contained alone, or both may be contained. One embodiment of the present invention is an antiallergic agent comprising a lotus crushed or extract or a combination thereof, wherein the antiallergic agent is a drug for improving or treating a disease caused by an allergic reaction. And preferably an agent for ameliorating and treating a disease caused by a type I allergic reaction. More preferably, the antiallergic agent of the present invention is a hay fever (in the present invention, hay fever refers to a symptom caused by an allergic reaction by pollen, and the main symptoms include, for example, runny nose, congested nose, and sneezing , Eye itch, sore throat, etc., but are not limited to these.), Dermatitis including atopic dermatitis, or bronchial asthma, or a combination or combination thereof For improving hay fever, dermatitis, atopic dermatitis or bronchial asthma. Still more preferably, the anti-allergic agent of the present invention is a hay fever improving agent. In another preferred embodiment, the antiallergic agent of the present invention is a serum IgE concentration-lowering agent for decreasing serum IgE concentration. Further, in another preferred embodiment, the antiallergic agent of the present invention is an IL-14 production inhibitor. In another preferred embodiment, the antiallergic agent of the present invention is a TNF production inhibitor.

The antiallergic agent of the present invention can be administered orally or parenteral administration such as intramuscular, intradermal, subcutaneous, intravenous, lower body cavity, skin, intranasal, oral or nasal suction administration. The drug of the present invention can contain a pharmaceutically acceptable component, and the component is a matter that can be recognized by those skilled in the art and is particularly limited. Not something. In order to formulate the antiallergic agent of the present invention, it is possible to formulate it by any ordinary method in the technical field of formulation.For example, in the case of oral administration, tablets, granules, powders, Dosage forms such as capsules, syrups, and troches can be employed. In the case of transdermal administration, ointments, patches, lotions, aerosols and the like can be mentioned. That is, when preparing a solid preparation for oral use, excipients and, if necessary, binders, lubricants, coloring agents, flavoring agents, etc. should be added to the crushed lotus extract or extract, or a combination thereof. After the addition, tablets, granules, powders, capsules, troches, sugar-coated tablets and the like can be made by a conventional method. The amount of the crushed lotus extract and the extract contained in the antiallergic agent of the present invention is not particularly limited as long as the antiallergic agent of the present invention exerts its effects.

 The dose of the antiallergic agent of the present invention to humans is as follows: Oral administration, for adults, per day, in the case of crushed material, 0 g, more preferably 2 g to 40 g. In the case of an extract, the amount is preferably 0.5 g to 50 g, more preferably 1 g to 20 g.

Another embodiment of the present invention is an anti-allergic food additive comprising a crushed or extracted lotus, or a combination thereof. The food additive in the present invention is not particularly limited as long as it is added to food. The food additive for anti-allergy herein is a food additive for improving and treating a disease caused by an allergic reaction to a person who has consumed food containing the food additive, and is preferably a type I food additive. It is a food additive for improving and treating diseases caused by allergic reactions. More preferably, the anti-allergic food additive of the present invention is a food additive for improving or treating pollinosis, dermatitis including atopic dermatitis, or bronchial asthma, or a combination thereof. A food additive for improving hay fever, a food additive for improving dermatitis, a food additive for improving atopic dermatitis, or a food additive for improving bronchial asthma. Even more preferably, the antiallergic food additive of the present invention comprises a flower It is a food additive for improving hay fever. In another preferred embodiment, the food additive for anti-allergic use of the present invention is a food additive for decreasing serum IgE concentration for decreasing serum IgE concentration. Further, in another preferred embodiment, the food additive for antiallergy of the present invention is a food additive for inhibiting IL-14 production. In another preferred embodiment, the food additive for anti-allergy of the present invention is a food additive for suppressing TNF production. In order to produce the antiallergic food additive of the present invention, it can be produced in the form of solids, granules, powders, capsules, solutions, suspensions, etc. by a usual method in the technical field of food additives. It is possible. The food additive of the present invention can include a component acceptable as a food additive, and the component is a matter that can be recognized by those skilled in the art and is not particularly limited.

 The amount of the crushed lotus extract and the extract contained in the antiallergic food additive of the present invention varies depending on the amount of the food additive added to the food, the type of the food, and the like, and is particularly limited. It is not done.

Another embodiment of the present invention is an antiallergic food comprising a lotus crushed or extracted product or a combination thereof, wherein the antiallergic food ameliorates a disease caused by an allergic reaction, A food for treatment, preferably a food for ameliorating and treating a disease caused by a type I allergic reaction. More preferably, the antiallergic food of the present invention is a food for improving or treating pollinosis, dermatitis such as atopic dermatitis, or bronchial asthma, or a combination thereof, that is, a food for improving hay fever. , A food for improving dermatitis, a food for improving atopic dermatitis, or a food for improving bronchial asthma. Still more preferably. The food for antiallergic use of the present invention is a food for improving hay fever. In another preferred embodiment, the food for anti-allergic use of the present invention is a food for decreasing serum IgE concentration for reducing serum IgE concentration. Further, in another preferred embodiment, the antiallergic food of the present invention is a food for suppressing IL-14 production. In another preferred embodiment, the antiallergic food of the present invention is a food for suppressing TNF production. The antiallergic food of the present invention is not particularly limited as long as it contains a crushed lotus extract or an extract thereof, or a combination thereof. The type of food is not particularly limited as long as it is usually consumed as food, and tablets, granules, powders, and capsules, which are called health foods, supplements, and dietary supplements Foods such as udon, buckwheat, pasta, ramen, etc., flour such as flour, buckwheat, potato starch, rice flour, baked goods such as confectionery bread and bread, cake , Cookies, rice crackers, anko, yokan, rice cake, dumplings, jelly and other confectionery, juices, tea and other beverages, instant foods, instant miso soup, instant soups and other instant foods. Not something. Preferably, the food of the present invention is a food containing crushed lotus and powders containing Z or extract, such as bread, cake, cookie, and rice cracker made from flour, buckwheat, potato starch, rice flour, and the like. It is. More preferably, the food of the present invention is a bread made from powdered crushed lotus and / or extract containing lotus, such as flour, buckwheat flour, potato starch, rice flour, etc. , Cakes, cookies, rice crackers, etc. In another preferred embodiment, the food of the present invention is a podart containing a ground lotus or extract of lotus, or a combination thereof. The yogurt has an advantageous effect of a synergistic effect between the effect of the crushed lotus and / or extract of lotus and the probiotic effect of lactic acid bacteria. The yogurt here includes not only semi-solid ordinary yogurt but also liquid yogurt such as yogurt drinks. Here, for the production of the food of the present invention described above, any known methods and materials can be used.

The amount of the crushed lotus extract and the extract contained in the antiallergic food of the present invention is not particularly limited as long as the antiallergic food of the present invention exerts its effects. The antiallergic food of the present invention is preferably used in the case of a human adult per day as a dry weight of a crushed lotus extract and an extract, in the case of a crushed product, preferably lg to 100 g, more preferably, It is in the form of food that consumes 2 g to 40 g, In the case of the extract, it is preferably in the form of a food to consume 0.5 g to 50 g, more preferably lg to 20 g.

 The antiallergic food of the present invention can be produced by adding a crushed lotus and _ or an extract to the raw material constituting the food, and the raw material constituting the food can be produced by adding the present invention. It can be produced by adding an antiallergic food additive. Further, depending on the type of food, an embodiment in which the antiallergic food additive of the present invention is added to the manufactured food to obtain the antiallergic food of the present invention is also possible. The oral drug, food additive and food of the present invention preferably contain lactic acid bacteria. In the present invention, oral drugs, food additives, and foods containing lactic acid bacteria can achieve the synergistic effect of the effect of the crushed lotus and Z or extract of lotus and the probiotic effect of the lactic acid bacteria. It is advantageous in terms of action. The lactic acid bacteria that can be used in this embodiment include the genus Lactobacillus, the genus Streptococcus, the genus Bifid ob acter i_um, and the genus Nocilas (Baci 11 us). Lactic acid bacteria belonging thereto, but are not particularly limited thereto. As a lactic acid bacterium, sporulated lactic acid bacterium is preferred from the viewpoint that the lactic acid bacterium easily reaches the intestine alive when taken orally. The spore-forming lactic acid bacterium is not particularly limited, but examples include Bacilluscoagulans.

Although not wishing to be bound by theory, the antiallergic effect of orally administered an antiallergic agent containing the crushed lotus extract or the extract of the present invention or a combination thereof to mammals including humans according to the present invention. One of the mechanisms of this is that oral administration of the antiallergic agent suppresses the production of interleukin 4 (IL-4) by type 2 helper T lymphocytes (Th2) in vivo, It is thought to be due to suppression of IgE production in cells. Another mechanism is thought to be due to suppression of the inflammatory reaction by suppressing the production of TNF in vivo, preferably by suppressing the production of TNF-a by oral administration of the antiallergic agent. 8

1 0 In addition, although the above-mentioned example was given as an action mechanism, this does not exclude other action mechanisms.

 The antiallergic agent, the antiallergic food additive and the antiallergic food of the present invention contain crushed lotus or extract of lotus, or a combination thereof as an active ingredient, so that hay fever, bronchial asthma, and atopic It has the advantageous effect of enabling the improvement and treatment of allergic diseases including dermatitis such as dermatitis. In addition, the drug, food additive and food of the present invention have an advantageous effect that the serum IgE concentration can be reduced by containing a crushed lotus extract or an extract thereof, or a combination thereof as an active ingredient.

 Furthermore, the drug, the food additive and the food of the present invention have an advantageous effect that the production of IL-4 in vivo can be suppressed by containing a lotus crushed product or an extract or a combination thereof as an active ingredient. Have.

 In addition, the drug, food additive and food of the present invention have an advantageous effect of being able to suppress TNF production in a living body by containing a crushed lotus or extract of lotus, or a combination thereof as an active ingredient.

 In addition, the drug, food additive and food of the present invention have an advantageous effect of being able to improve and treat coldness by containing a crushed lotus extract or extract or a combination thereof as an active ingredient. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing the serum IgE concentration of each subject before the start of ingestion of a lotus root extract (week 0) and at the 9th week of ingestion in Example 2.

 FIG. 2 is a graph showing the serum IgG concentration of each subject before the start of ingestion of a lotus root extract (week 0) and at 9 weeks of ingestion in Example 2.

FIG. 3 is a graph showing the IL-12 concentration in the lymphocyte culture supernatant for each subject before the start of the intake of the lotus extract (week 0) and at the eighth week of the intake in Example 2. FIG. 4 is a graph showing the IL-14 concentration in the lymphocyte culture supernatant for each subject before starting the intake of the lotus root extract (week 0) and at 6 to 8 weeks of the intake in Example 2. .

 FIG. 5 is a graph showing variation of IgE in a pollen subject in Example 3.

 FIG. 6 is a graph showing variation of IgE in a pollen subject in Example 3. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the scope of the examples.

Example

 Example 1: Effect of oral ingestion of lotus root extract in mice administered with cedar pollen

 1) Preparation method of lotus root extract

 20 g of commercially available lotus root was cut into small pieces, 5 OmL of distilled water was added thereto, and the mixture was boiled for 8 minutes, filtered, and the filtrate was used as a lotus root extract.

 2) Oral administration of lotus root extract and nasal administration of cedar pollen extract

 Seven days before administration of the cedar pollen extract, mice (BAL BZc; female, 3-week-old) were orally administered 0.5 mL of a lotus root extract per animal once a day using a sonde (pre-administration). ). The mice were bred in a cycle of turning on for 12 hours and turning off for 12 hours while allowing them to freely take feed (a commercially available solid feed) and tap water. After the above pre-administration for 7 days, the Lotus root extract is orally administered once a day for another 14 days (this administration), and in parallel with the Japanese cedar pollen extract (Torii Pharmaceutical; Japanese cedar pollen allergen for scratching) Was instilled into the nasal cavity of the mouse with a micropipet to artificially produce a cedar pollen-sensitized mouse (Example 1).

As Comparative Example 1, the same amount of distilled water was administered instead of the lotus root extract. Was set as a control group. The experiment used 9 mice in each group.

 3) Serum collection method

 Four hours after the last administration for 14 days, the mice were anesthetized with ether, and blood was collected from the hepatic artery using a 1 mL syringe. After leaving at 37 ° C for 60 minutes, serum was separated.

 4) Serum IgG measurement by SR ID method (in-plate simple immunodiffusion method)

 The serum IgG concentration was measured by the SR ID method. The measurement was performed according to the method described in Example 2 described later.

 5) Serum IgE measurement by ELISA (enzyme-linked immunosorbent assay)

 The IgE concentration in the serum was measured by the ELISA method. The measurement was performed according to the method described in Example 2 described later.

 6) Culture of peritoneal macrophages and measurement of TNF-α production

 Two days before the collection of peritoneal macrophages, 1 mL of thioglycolate was injected intraperitoneally. Prior to blood collection on the day of the experiment, 3-5 mL of cold MEM was injected, and macrophages were collected (repeated 3-4 times).

The cell concentration was adjusted to 2 × 10 ⁶ ce 11 ZmL, suspended in MEM containing 10 g LPS (from Escherichia coli), and placed in a 12-well plate at a volume of 1 mL and cultured at 37 ° C. for 24 hours. This culture supernatant was collected and stored at 180 ° C until measurement.

 TNF— was measured by an ELISA method using a kit manufactured by Wako Pure Chemical Industries.

7) Culture of spleen cells and measurement of IL-2 and IL-4 production

 At the time of blood collection, the spleen is aseptically removed, a 200-mesh metal filter for cells is placed in a small Petri dish containing 2 mL of RPMI 1640 culture solution, the spleen extracted is placed on the filter, and the piston push face of the 5 mL syringe is placed. The spleen cells were removed from the spleen by using.

The obtained spleen cells were finally suspended in RPMI 1640 containing 10% FBS and 20 g ConA (concanapalin A) so as to have l × 10 ⁶ cel 1 / mL. Then, add the cell suspension to the 12-well plate by lmL, They were cultured for 48 hours in 5% C0 ₂ presence. The culture supernatant was stored at -80 ° C until measurement.

 IL-1 and IL-4 were measured by the ELISA method. The measurement was performed according to the method described in Example 2 below.

 The serum IgG was 469 mg ZdL in the control group, Comparative Example 1, whereas it was 875 mg / dL in Example 1, which was the lotus root extract-administered group, about 1.86 times that of the comparative example. . This suggests that lotus root extract increases the ability to protect against infection by increasing IgG in serum.

 In contrast, IgE, which is closely related to type I allergy, was 87.2 ng / dL in the comparative example, whereas 38.6 ng / dL in Example 1 Was about 0.44 times lower than

 From these results, it was clarified that oral administration of a lotus root extract can increase serum IgG and reduce serum IgE.

 Regarding TNF-α production by macrophages, the concentration of TNF-α in the culture solution in Comparative Example 1 was 37.2 pgZmL, whereas in Example 1, it was 15.5 pg / mL, which was about 0%. It had dropped 42 times.

 From this, it is clear that oral administration of a lotus root extract suppresses the production of TNF-hi, and one of the mechanisms of action of the anti-allergic action in the present invention may be the inhibition of TNF production. It is inferred.

 Regarding IL-12 production of spleen cells, the concentration of IL-12 in the culture solution in Comparative Example 1 was 63.6 pgZmL, whereas that in Example 1 was 94.2 pg mL, which was about 1. Had increased 48-fold. This suggests that the lotus root extract enhances cellular immunity induced by ΤΙΊ1.

In contrast, the amount of IL-14 involved in IgE production in 生 体 cells in vivo was 116.4 pgZmL in Comparative Example 1, whereas 36.8 pg / mL in Example 1. mL, which was about 0.32 times lower. From this, it became clear that oral administration of a lotus root extract promoted the production of IL-12 and suppressed the production of IL-4.

 From Example 1 and Comparative Example 1, the oral administration of a lotus root extract promoted the production of IL-12 in vivo, while suppressing the production of IL-4 in vivo, and It is presumed that IgE in the medium is reduced, which may cause an antiallergic effect.

 Example 2: Effect of continuous intake of lotus root extract on human immune system

 1) Preparation method of lotus root extract

 100 kg of commercial lotus root was peeled, washed with water, and sliced to a thickness of 5 to 10 mm. 260 L of water was added, water and lotus root were charged into a kneader, the temperature was raised to 98 ° C, and the mixture was stirred. After heating to 99t :, the mixture was boiled for 30 minutes. Next, the boiled product was taken out from the kneader, and the filtrate obtained by filtration through salad was powdered by freeze-drying. By this operation, 5.0 kg of a lotus root extract powder was obtained. An equal amount of lactose was added to 5.0 kg of this powder to prepare 10.0 kg of round white tablets (approximately 250 mgZ) having a diameter of 8 mm, which was designated as an antiallergic agent LRE.

 2) Dosage and administration schedule of lotus root extract

 Four women (21 years old, pollen quality) were the subjects.

 Subjects ingested LRE twice a day in the morning and in the evening, with eight tablets of water or hot water before meals. If you forgot to take it in the morning, it was decided to take a double dose before dinner. LRE was taken for 9 weeks. '

3) Collection of serum and lymphocytes in peripheral blood from subjects

 Serum and lymphocytes were collected and used for experiments before the start of continuous ingestion of the lotus root extract and every seven days after ingestion in the following manner. All operations were performed aseptically, and sterilized instruments were used.

a) 1 OmL of venous blood was collected, and 2 mL was dispensed into blood collection tubes for serum separation, and the remaining 4 mL was dispensed into two blood collection tubes containing an anticoagulant (sodium citrate). b) After leaving the blood collection tube for serum separation at 37 ° C for 1 hour, perform centrifugal operation at 3000 rpm for 15 minutes, take the obtained serum into a microtube, and freeze at 180 ° C until use. saved.

 c) In order to obtain lymphocytes from peripheral blood, blood collected in a blood collection tube containing an anticoagulant was diluted 2-fold with PBS (pH 7.2). This was overlaid on human lymphocyte separation solution (LSM) having a specific gravity of 1.077, and subjected to refrigerated centrifugation at 1600 rpm for 20 minutes to obtain total lymphocytes at the interface.

 d) Lymphocytes were collected in a small test tube, washed twice with PBS by cooling at 1400 rpm for 10 minutes twice. When erythrocytes were mixed, the erythrocytes were hemolyzed by leaving them to stand at 37 ° C for 10 minutes using filter-sterilized ammonium tris chloride.

e) The supernatant was removed, and ImL of RPMI 1640 culture solution containing 20 g ZmL of Concanapalin A (Con A) was added. The mixture was mixed well, and the cell number was determined using a Thai-style blood counter. The cell number was adjusted to 2 × 10 ⁶ ce 1 1 / mL. Thereafter, the lymphocyte suspension was cultured at 37 ° C for 48 hours using an 8-well plate for culture.

 f) After the culture, the supernatant was collected in a microtube, centrifuged at 6000 rpm for 5 minutes, and the supernatant was transferred to another microtube and stored frozen at 180 until the day of the experiment.

 4) Measurement of serum IgE by ELISA

 The quantification of IgE antibody present in the serum of the subject is performed by the following ELISA.

 / This.

 a) Anti-I ^ £ antibody 5 diluted 100-fold with sodium carbonate buffer was added to 100, and 100 were added to a well of a 96-well plate for ISA, and immobilized at room temperature for 60 minutes.

 b) After washing twice with a washing solution, 200 μL of a buffer for phosphate—coat was added, and the mixture was incubated at 37 ° C. for 30 minutes.

C) After washing twice with washing solution, serum of subject and IgE standard solution (1.87; 3.7) 5; 7.5; 15; 30 ng / mL) and reacted at 37 ° C for 60 minutes.

 d) After washing twice with the washing solution, 100 L of peroxidase (HRP) standard antibody diluted 2000-fold was added, and reacted at 37 ° C for 60 minutes.

 e) After washing three times with the washing solution, 100 L of a coloring solution (o-phenylenediamine solution; OPD solution) was added, and reacted at room temperature for 30 minutes.

 f) After color development, stop solution (3 M sulfuric acid) was added in lOO 3 ^ L, and the absorbance at 490 nm was measured with an ELISA reader.

 g) The IgE concentration in the serum of the subject was calculated from the standard curve.

 FIG. 1 shows a graph of the serum IgE concentration before the start of the intake of the lotus extract (week 0) and at the 9th week of the intake in each subject.

5) Measurement of serum IgG by SR ID method

 The IgG antibody present in the serum of the subject was quantified by the following SRID method.

 a) Using a microsyringe well washed with physiological saline, aliquots of standard IgG serum (543, 1604, 3053 mg ZdL) and the serum of each subject were exactly 4 in agarose plates for MBL human IgG quantification (medical biotechnology). Research Institute). The plate was covered and incubated at room temperature for 24 hours. b) After the reaction, the diameter of the resulting sedimentation ring was measured, and the IgG concentration in the serum of the subject was calculated from the standard curve.

 FIG. 2 shows a graph of serum IgG concentrations before the start of the intake of the lotus extract (week 0) and at the 9th week of the intake for each subject.

 6) Measurement of IL-12 in lymphocyte culture supernatant by ELISA

 The quantification of IL-12 secreted from peripheral blood lymphocytes cultured for 48 hours was performed by the following ELISA.

a) Serum of subject and IL-2 standard solution in a well of a 96-well plate for ELISA (31, 25, 62.5, 125, 250, 500, 1000 pg / mL) were added in 100 iiL.

 b) 50 L of a biotinylated antibody (anti-IL-12) was added, and the mixture was incubated at room temperature for 1 hour.

 c) After washing three times, 100 L of streptavidin-HRP was added and reacted at room temperature for 30 minutes.

 d) After washing three times, 100 parts of color developing solution (TMB) was added, and the reaction was carried out at room temperature for 10 to 15 minutes while protecting from light.

 e) 100 parts of stop solution (sulfuric acid) was added, and the absorbance at 450 nm was measured with an ELISA reader.

 f) The IL-12 concentration in the test subject lymphocyte culture supernatant was calculated from the standard curve. FIG. 3 shows a graph of the IL-12 concentration in the lymphocyte culture supernatant before the start of the intake of the lotus root extract (week 0) and at the 8th week of the intake in each subject.

 7) Measurement of IL-14 in lymphocyte culture supernatant by ELISA

 IL-4 secreted from peripheral blood lymphocytes cultured for 48 hours was quantified by the following ELISA.

 a) 100 wells of the serum of the subject and IL-14 standard solution (1.21, .4, 8.75, 17.5, 35 pg / mL) were placed in a well of an ELISA 96-well plate, and incubated at room temperature for 2 hours. .

 b) After washing three times, 50 L of a biotinylated antibody (anti-IL-4) was added, and incubated at room temperature for 1 hour.

 c) After washing three times, 100, "L of streptavidin-HRP was added and reacted at room temperature for 30 minutes.

 d) After washing three times, 100 L of a color developing solution (TMB) was added, and the reaction was carried out at room temperature for 15 to 20 minutes while protecting from light.

e) Add stop solution (sulfuric acid) at 100 / L and read at 450 nm with an ELISA reader. Was measured for absorbance.

 f) From the standard curve, the IL-14 concentration in the subject's lymphocyte culture supernatant was calculated. FIG. 4 shows a graph of the IL-14 concentration in the lymphocyte culture supernatant before the start of the intake of the lotus root extract (week 0) and between 6 and 8 weeks of the intake for each subject.

 In Example 2, ingestion of LRE, an anti-allergic agent containing lotus root extract, for 9 weeks reduced the serum IgE concentration in all four subjects compared to before LRE intake. Was observed. In contrast, an increase in serum concentration of IgG was observed in all four subjects.

 In addition, ingestion of LRE, an extract of lotus root, for 8 weeks increased the production of IL-12 in peripheral blood lymphocytes in all four subjects, compared to before ingestion of LRE. Admitted. On the other hand, regarding the production of IL-4, the intake of LRE for 6 to 8 weeks suppressed the production of IL-14 in peripheral blood lymphocytes in all four subjects. Was recognized. From Example 2, it can be seen that oral intake of a lotus root extract promotes the production of IL-12 in vivo and suppresses the production of IL-14 in vivo, thereby increasing the IgE in serum. It is presumed that anti-allergic effects can occur.

 Example 3: Effect of continuous intake of lotus root extract on improvement of pollen quality

 1) Preparation method of lotus root extract

 The antiallergic agent LRE containing the lotus root extract used in Example 2 was used.

 2) Dosage and schedule of lotus root extract

17 flower powdery subjects, 2 males (1 in their 40s, 1 in their 30s), 15 females (3 in their 50s, 5 in their 40s, 1 in their 30s, 3 in their 20s, 3 in their 10s) ) And two subjects without pollen (1 male in their 50s and 1 female in their 40s) for a total of 19 subjects. Subjects consumed LRE twice daily in the morning and evening before meals with water or hot water. If you forgot to take in the morning, you should take twice the dose before dinner. LRE was taken for three months (from early February to early March).

 3) Collection of serum from the subject

 Before the start of continuous ingestion of the lotus root extract and about once a month after ingestion, blood is collected at a hospital, the serum is separated, and serum IgE (non-specific IgE), cedar pollen-specific IgE and cypress are obtained. The pollen-specific IgE concentration was measured. These measurements were performed by PLM Corporation.

 For pollen-specific IgE, the IgE concentration was evaluated in seven stages from 0 to 6. Class 0: 0.34 (UA / HIL, hereinafter the same unit) Class 1: 0.35 to 0.69, Class 2: 0.70 to 3.49, Class 3: 3.5 0-17.49, Class 4: 17.5-49.9, Class 5: 50.0-99.9, Class 6: 100 or more.

 4) Improvement of hay fever symptoms

 The degree of improvement of hay fever symptoms was determined by the allergy symptoms (sneezing, runny nose: stuffy nose, itchy eyes, foreign eyes, foreign body sensation, eyelid discomfort, other hot flashes, headache, skin itch during the test period. , Pharyngeal itching, mouth itching, respiratory abnormal sounds, etc.) The subjects improved their symptoms in four stages (no improvement; mild improvement; moderate improvement; You must declare yourself.

 The results of Example 3 are shown below.

Effect of pollen on serum IgE (non-specific IgE) in subjects There were 14 (73.7%). Of these, 40% reduction is 2 people (10.5%; male 30s, 1 female, 1 female 40s), 30% reduction is 7 (36.8%; male 40s, 1 female, 5 women) 0s 1 person, 40s 3 people, 20s 1 person, 1 0 in 1 generation), 20% reduction in 4 (21.0%; 1 female 50s, 2 in 20s, 1 teenage), 1% reduction in 1 (5.3%; female 50s) ) Met. One (5.3%; women in their 40s) had no change at all, while 2 (10.5%; women in their thirties and one in their teens) had increased but hardly changed. there were. As described above, the effect of the antiallergic agent LRE was observed, but this effect was not observed depending on age or gender.

 As a representative example, graphs of changes in IgE concentration in subjects with decreased IgE concentration are shown in Figure 5 (male 30s) and Figure 6 (female 20s). 5 and 6, the vertical axis represents the IgE concentration (non-specific IgE concentration) and the pollen-specific IgE, and the horizontal axis represents the blood collection date. In Figure 5, the test start date is December 4, and in Figure 6, the test start date is December 5.

 Effect of pollen quality on serum pollen-specific IgE in subjects

 In pollen-type subjects, an increase in the concentration of non-specific IgE increases the concentration of pollen-specific IgE, and a decrease in the concentration of non-specific IgE tends to decrease the concentration of pollen-specific IgE. Was done. FIG. 6 also shows that the cedar-specific IgE and the hinoki-specific IgE were reduced along with the decrease in non-specific IgE.

 Effects on non-pollinated subjects

 Non-pollinated two subjects (one male in their 50s and one female in their 40s) both showed a decrease in non-specific IgE levels. Three months after the start of the test, the degree of the decrease was 39., 37 IU / mL, and 8 to 5 IUZmL, respectively. The pollen-specific IgE was class 0 at any time during the test period.

 Evaluation of improvement of allergic symptoms in pollen subjects

Seven of the 17 flower powdery subjects (41.2%) had improved allergic symptoms compared to the previous year. Also, during the three-month test period, no serious physical trouble was observed due to ingestion of the antiallergic agent LRE, and it was confirmed that there were no dangerous side effects. In 5 subjects, the symptoms of dermatitis including rough skin were improved by administration for 3 months.

 In seven subjects, improvement in coldness due to subjective symptoms was observed after administration for 3 months.

 Example 4: Effect of continuous intake of lotus root extract on improvement of severe flower powder quality

1) Preparation method of lotus root extract

 The antiallergic agent LRE containing the lotus root extract used in Example 2 was used.

 2) Dosage and administration schedule of lotus root extract

 18 subjects with severe flower powder quality compared to the subjects of Example 3 [7 males (1 male in 50s, 4 males in 40s, 1 male in 30s, 1 male in 20s), female 11 (2 people in their 40s, 6 people in their 30s, 3 people in their 20s)] were used as test subjects. Severity was judged based on the self-report of the subject.

 Subjects consumed LRE twice daily in the morning and evening before meals with 11 tablets of water or hot water. If you forgot to take it in the morning, double the amount before dinner. LRE intake occurred for about 5 months (from early February to late April).

 3) Improvement of hay fever symptoms

 The degree of improvement of the symptoms of hay fever was determined by the allergy symptoms (sneezing, runny nose, stuffy nose, itchy eyes, irritated eyes, foreign eye sensation, eyelid discomfort, other hot flashes, headache, skin itch during the test period. , Pharyngeal itching, mouth itching, abnormal breathing, etc.) in four stages (no improvement; mild improvement; moderate improvement; remarkable improvement) Subjects self-reported.

The results of Example 4 are shown below. Evaluation of amelioration of allergic symptoms in pollen subjects

 Allergic symptoms were significantly improved in 5 subjects (18%), moderately improved in 7 subjects (39%), and mildly improved in 18 allergic symptoms in 18 pollen subjects Four subjects (22%) had no improvement, and two subjects (11%) did not improve. 89% of all subjects showed improvement in allergic symptoms compared to the previous year. The two subjects who did not improve had discontinued LRE in about one month. Considering this point, all the subjects with pollen quality who continued taking LRE showed improvement in allergic symptoms.

 In Example 4, the degree of improvement in allergic symptoms was better than that in Example 3. Although the reason for this is not clear, the possible causes include the fact that the intake of the anti-allergic agent LRE was large in Example 4 and the degree of pollen quality of the subject was severe.

 Also, during the test period of about 5 months, no serious physical trouble was observed due to the ingestion of the antiallergic agent LRE, and it was confirmed that there were no dangerous side effects. Industrial applicability

 The drug, food additive and food of the present invention can be used for antiallergy, amelioration of hay fever, reduction of serum IgE concentration, suppression of IL-14 production, and suppression of TNF production.

Claims

The scope of the claims 1. An anti-allergic agent containing crushed or extracted lotus, or a combination thereof.  2. The anti-allergic agent according to claim 1, wherein the crushed lotus extract or extract or a combination thereof is a crushed lotus root extract or an extract thereof or a combination thereof.  3. Food additives for anti-allergy containing crushed or extracted lotus, or combinations thereof.  4. Anti-allergic foods containing crushed or extracted lotus, or combinations thereof.  5. The antiallergic food according to claim 4, wherein the crushed lotus extract or extract or a combination thereof is a crushed lotus root extract or an extract thereof or a combination thereof.  6. Hay fever containing crushed or extracted lotus products or a combination of these 7. Food additives for improving hay fever, including crushed or extracted lotus products or a combination thereof.  8. A hay fever-improving food containing broken lotus or extract of lotus, or a combination thereof.  9. A serum IgE concentration-lowering agent containing a lotus crush or extract or a combination thereof.  10. A food additive for lowering serum IgE concentration, including crushed lotus extract or extract, or a combination thereof. 11. A food for reducing serum IgE concentration, which contains broken lotus or extract of lotus, or a combination thereof. 12. An IL-14 production inhibitor containing a broken lotus or extract of lotus, or a combination thereof.  13. A food additive for inhibiting the production of IL-14, comprising a crushed or extracted lotus plant or a combination thereof.  14. A food for inhibiting production of IL-14, comprising a broken lotus or extract of lotus, or a combination thereof.  15. A TNF production inhibitor comprising a crushed or extracted lotus plant or a combination thereof.  16. A food additive for suppressing TNF production, comprising a crushed or extracted lotus plant or a combination thereof.  17. A food for suppressing TNF production, comprising a crushed or extracted lotus plant or a combination thereof.  18. A food product selected from the group consisting of bread, cake, cookery and rice crackers, manufactured from flour containing crushed or extracted lotus products or a combination thereof.  19. A dart containing crushed or extracted lotus, or a combination thereof.  20. An instant food selected from the group consisting of instant 麵, instant miso soup and instant soup, comprising a lotus crush or extract or a combination thereof.  21. Contains lots or extracts of lotus, or combinations thereof.