JPWO2005092357A1 - Antiallergic and anti-inflammatory agents - Google Patents

Abstract

The present invention relates to an antiallergic agent, anti-inflammatory agent, basophil degranulation inhibitor, mast cell degranulation inhibitor, or β-hexosamini containing a Lycium plant, or a polar solvent extract thereof. It relates to an inhibitor of oxidase release. The wolfberry plant is a natural product with dietary experience, and can provide a highly safe antiallergic agent and antiinflammatory agent by a simple method. Moreover, the antiallergic agent and anti-inflammatory agent of this invention can also be mix | blended with food-drinks, cosmetics, a bath agent, a pharmaceutical, etc.

Description

  The present invention relates to an antiallergic agent and an anti-inflammatory agent that can be used as food or drink for humans or animals, pharmaceuticals, and cosmetics.

  The living body originally has an immune function for excluding foreign substances from outside and maintaining homeostasis. However, this immune function may work to damage the body, and this kind of disorder reaction due to excessive immune function is called allergy. Allergies are caused by excessive reaction of immunocompetent cells with causative substances derived from microorganisms, plants, animals, drugs, chemical substances, foods, etc., and activated basophils, mast cells, T lymphocytes, B lymphocytes, etc. The tissue in the body is damaged by the physiologically active substance released from the body. Depending on the time from reaction to onset, the allergic reaction may occur in a relatively short period of time from several minutes to several tens of minutes. Divided into delayed allergies.

  Basophils present in the blood and mast cells present in the vicinity of blood vessels and connective tissues have granules containing histamine, etc., and these cells degranulate, thereby causing enzymes such as β-hexosaminidase along with histamine. It is known to liberate and induce immediate allergic reactions such as asthma and atopic eczema.

  Allergic diseases are not the same as atopic diseases. Allergic factors are involved in some atopic diseases. However, as represented by atopic dermatitis, microbial infections and genetic factors also play a complex role in the onset of atopic diseases. .

  In addition, substances released by degranulation of basophils and mast cells are well known as substances that cause inflammation.

  Thus, mast cells and basophils are strongly involved in immediate allergy, and it is considered that allergy can be reduced by inhibiting degranulation of these cells. Therefore, it is desired to develop an effective basophil cell degranulation inhibitor or mast cell degranulation inhibitor to provide a useful antiallergic agent.

  Antiallergic agents include drugs that suppress degranulation and release of chemical mediators from mast cells in type I allergic reactions, which are immediate allergies, drugs that suppress the binding of histamine and H1 receptors, and lipid-type chemicals. Drugs that suppress the mediator thromboxane A2 and drugs that suppress leukotrienes have been developed. However, there are side effects such as drowsiness and sedation, central nervous system inhibitory effects, and cystitis-like symptoms such as hematuria. Absent. In particular, anti-allergic agents are often used for preventive purposes not only for treatment but also for their use, and their safety is particularly important.

  Lycium plant is a solanaceous plant, its leaves are called cocoon leaves, fruit is called coconut, root bark is called skeletal skin, liver function strengthening action, hypertension prevention, arteriosclerosis prevention, presbyopia prevention, etc. It is said to have the action of.

  In addition to carotenes and vitamins, wolfberry plants contain β-sitosterol, linoleic acid, zeaxanthin, betaine and fezarien. As a pharmacological effect, it has been reported that betaine has an effect on lipid metabolism and anti-fatty liver, and the anti-fatty liver action of the aqueous extract on the liver is known.

  Regarding the anti-allergic effect of wolfberry, 5-lipoxygenase activity inhibitors containing an easily soluble component in a low-polar organic solvent and an anti-allergic food containing the same have been reported. Antiallergic action is not recognized (Patent Document 1).

In addition, the effect of wolfberry plants on atopic disease has been reported, but it refers to the effect on atopic dermatitis, and is attributed to its basophil cell degranulation inhibitory activity or mast cell degranulation inhibitory activity. No antiallergic action has been found (Patent Document 2). Furthermore, there has been no report that a wolfberry plant or its polar solvent has basophil cell degranulation inhibitory activity, mast cell degranulation inhibitory activity, or β-hexosaminidase release inhibitory activity.
JP 2001-233778 A JP 2004-67526 A

  An object of the present invention is to provide a new antiallergic agent and an anti-inflammatory agent that can be easily manufactured with high safety and can be blended in cosmetics and foods and drinks.

  As a result of intensive studies to solve the above problems, the present inventors have found that an extract obtained by extracting a Lycium plant with a polar solvent has a β-hexosaminidase inhibitory activity, a basophile. It has been found that it has sphere cell degranulation inhibitory activity and mast cell degranulation inhibitory activity, and has antiallergic action and anti-inflammatory action, and has completed the present invention.

  That is, the present invention relates to an antiallergic agent containing a Lycium plant or an extract thereof as an active ingredient.

  The present invention also relates to a basophil cell degranulation inhibitor containing a Lycium plant or an extract thereof.

  The present invention also relates to a mast cell degranulation inhibitor containing a Lycium plant or an extract thereof.

  The present invention also relates to a β-hexosaminidase release inhibitor characterized by containing an extract of a Lycium plant.

  The antiallergic agent and anti-inflammatory agent of the present invention may be either a preventive agent or an improving agent, and of course may also serve as both.

The wolfberry extract shown in this specification is useful as an antiallergic agent and an anti-inflammatory agent. The wolfberry extract of the present invention is highly safe and can be prepared by a simple method. Therefore, the wolfberry extract is expected to be highly applicable to pharmaceuticals, foods and drinks, cosmetics, bath preparations, etc. Is done.

It is a figure which shows the inhibition rate with respect to (beta) -hexosaminidase release of the dilution ratio of a wolfberry hot water extract, and a human basophil.

  Hereinafter, embodiments of the present invention will be described in detail.

  As mentioned above, the genus Lycium is a plant of the solanaceous family, and there are types such as Lycium chinense (sometimes referred to as Lycium chinensis), Lycium barbarum, Lycium turcomanicum, Lycium potaninii, Lycium dasystemum, etc. . The plant of the genus Arborella used for the antiallergic agent, antibasophil cell degranulation inhibitor, mast cell degranulation inhibitor or β-hexosaminidase release inhibitor of the present invention is not particularly limited as long as it is genus Octopus. chinense or Lycium barbarum is preferred, and Lycium chinense is more preferred.

  The production method of the genus Lycium used in the present invention is not particularly limited. The wolfberry plant used in the present invention is not limited to a native plant, but may be an artificially cultivated plant. In addition, there is no particular limitation on the collection time, the growing year, the cultivation method, and the cultivation period of the wolfberry plant used in the present invention.

  The wolfberry plant used in the present invention may be used by selecting only one kind from among various production methods, or may be used in combination of plural kinds.

  The extract of the wolfberry plant is obtained by extracting various parts (whole grass, flowers, leaves, etc.) of the plant body as they are or after pulverization and then extracting them with an extraction solvent. The part used for extraction is not particularly limited, but leaves (bamboo leaves), fruits (coconuts), root barks (earthbone barks) are preferred, and fruits (coconuts) are more preferred.

  A polar solvent can be used as the extraction solvent. Examples of polar solvents include polar organic solvents such as water or alcohols (eg, ethanol, lower alcohols of methanol, or polyhydric alcohols such as propylene glycol), and these may be used alone or in any combination of two or more. Can be used. Preferred is water alone or a mixed solvent of water and a polar organic solvent.

  In the case of a mixed solvent of water and a polar organic solvent, the mixing ratio of water and the polar organic solvent is not particularly limited, but water is the main component, that is, the volume ratio of water is preferably 50% or more. The polar organic solvent to be mixed with water is preferably a lower alcohol, and more preferably methanol or ethanol.

  When extracted with water as the main component, the extracted components are different because the polarity as the solvent is different from that when the organic solvent is used as the main component. The amount of the extraction solvent to be used is preferably 0.5 to 50 wt.

  In addition, the extraction method is not particularly limited, but it may be usually performed using an extraction solvent at normal temperature and normal pressure. In this case, the extraction time is not particularly limited, but 2 hours to 14 days is preferable. After extraction, it may be in the form of a solution, paste, gel, or powder by concentration to dryness or oil or fat. Depending on the case, extraction under conditions of 30 to 120 ° C. or extraction under supercritical conditions such as autoclave or carbon dioxide is possible, and the extraction time may be set appropriately. If necessary, it can be further purified by an arbitrary operation using an activated carbon column, an ion exchange resin, or the like.

  As one embodiment of the present invention, an extract obtained by extracting a wolfberry plant with hot water can be used. It is preferable that the amount of hot water used for the extraction is an amount that can sufficiently dissolve the active ingredient contained in the wolfberry plant.

  In the present invention, the wolfberry plant is extracted after washing the collected wolfberry plant to remove dirt and organisms adhering to the surface, and then crushing it by cutting, ball milling, ultrasonic treatment, homogenizer, etc. can do. Prior to crushing, air drying or freeze drying may be performed. The crushed wolfberry plant is preferably extracted several times with an extraction solvent such as hot water while stirring. The extract can be used as it is as a basophil cell degranulation inhibitor, mast cell degranulation inhibitor, β-hexosaminidase release inhibitor, antiallergic agent or anti-inflammatory agent.

  The wolfberry plant extract of the present invention can be adjusted to an appropriate concentration by diluting or concentrating as necessary. For example, it is possible to dilute the extract of the present invention by adding water or alcohol to use as a water-solubilized product, or to mix with oils and fats to make a cream. Furthermore, the basophil cell degranulation inhibitor, mast cell degranulation inhibitor, β-hexosaminidase release inhibitor, antiallergic agent or anti-inflammatory agent is obtained by spray drying the extract. You can also. Thus, the concentration and shape of the extract of the present invention are not particularly limited, and can be appropriately determined according to the application.

  The extract of the present invention may contain components other than the extract of the wolfberry plant. Since the extract of the present invention uses an extract of a wolfberry plant used for food as an active ingredient, it is highly safe for a living body. For this reason, the extract of the present invention can be contained in a wide range of products such as pharmaceuticals, functional foods, foods and drinks, quasi drugs, cosmetics, and bath preparations.

  The wolfberry plant or the wolfberry plant extract of the present invention has a β-hexosaminidase release inhibitory activity, and therefore has a degranulation inhibitory effect from basophils or mast cells. Therefore, β-hexosaminidase It can be used as a release inhibitor, a basophil cell degranulation inhibitor or a mast cell degranulation inhibitor, and is also useful as an antiallergic agent or antiinflammatory agent. That is, β-hexosaminidase is one of substances released from basophils or mast cells with degranulation. Therefore, by measuring β-hexosaminidase released by degranulation from cells by β-hexosaminidase release assay, it is possible to evaluate antiallergic action and anti-inflammatory action.

  In the present invention, basophil cell degranulation inhibitory activity or mast cell degranulation inhibitory activity is measured by β-hexosaminidase activity released by degranulation. β-hexosaminidase activity was released using basophil cells such as KU812 shown in Example 3 to be described later and sensitized with mouse anti-DNP-IgE antibody, and then released using antigen DNP-BSA. Measured β-hexosaminidase activity. In addition, mast cells may be used for measuring β-hexosaminidase activity. If wolfberry is used in combination with basophil cell degranulation inhibitory activity or mast cell degranulation inhibitory activity, stronger antiallergic and anti-inflammatory effects can be expected.

  The anti-allergic agent and anti-inflammatory agent of the present invention comprises a wolfberry plant or an extract of a wolfberry plant as an active ingredient, and may be an extract or an extract itself, or a known carrier or auxiliary, It may be a composition to which a material, other pharmaceutically acceptable formulation material, or the like is added. The blending amount is not particularly specified, but the concentration of the extract of the present invention in these products can be appropriately selected within a range that exhibits a desired effect. Moreover, as a dosage in the case of administering to a human body, it can be divided into 1 to several times, for example, as 0.2-100 mg / Kg body weight per day. The dosage form in the case of pharmaceutical preparations is tablets, capsules, injections, drops, powders, suppositories, granules, ointments, suspensions, emulsions, syrups, depending on the purpose and route of administration. It can be made into a cream or the like.

  The composition may contain additives such as binders, excipients, lubricants, disintegrants, stabilizers, emulsifiers, and buffers that are generally used in formulations. Preferable examples of the binder include starch, trehalose, dextrin, gum arabic powder and the like. Preferable examples of the lubricant include stearic acid, talc, sucrose fatty acid ester, polyethylene glycol and the like. Preferable examples of the disintegrant include starch, carboxymethyl cellulose, corn starch and the like. Preferable examples of the stabilizer include fats and oils and propylene glycol. Preferable examples of the emulsifier include anionic surfactants, nonionic surfactants, polyvinyl alcohol and the like. Preferable examples of the buffer include buffers such as phosphate, carbonate and citrate.

  Furthermore, the antiallergic agent or anti-inflammatory agent of the present invention can be contained in foods and drinks, cosmetics or bath preparations, and the content thereof is not particularly specified, but is usually 0.0001% by weight or more, preferably 0.01 to 100% by weight. good. When the extract of the present invention is contained in cosmetics and used as an antiallergic agent or antiinflammatory agent, it can be made 0.01 to 10% by weight.

    The anti-allergic agent or anti-inflammatory agent of the present invention can be made into a functional food for the purpose of preventing and / or improving allergy and inflammation by including in the food. The kind of food used as object will not be specifically limited if the antiallergic action and anti-inflammatory action of an extract are not inhibited. For example, juices, soft drinks, beverages such as tea, processed foods such as bread and rice cakes, sweets such as candy, instant foods such as cup noodles, butters, fats and oils such as salad oil, dressings, mayonnaise, sauces, soy sauce and so on It can be included in a wide range of foods and drinks such as seasonings such as mirin, sprinkles and miso. Of course, instead of the extract, wolfberry plants can be pulverized as they are and directly taken in addition to foods or the like.

  Hereinafter, the present invention will be described more specifically by describing production examples, examples and formulation examples. However, the scope of the present invention is not limited to these examples.

(Example 1)
After cutting and crushing 1 g of dry weight of coconut (derived from Lycium chinense), 10 ml of Milli-Q water was added and autoclaved at 105 ° C. for 15 minutes. Centrifugation was performed at 4 ° C. and 3000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter to obtain a hot water extract sample. Further, the extract was diluted 100-fold, 1000-fold, and 10000-fold with a cell culture medium.

(Example 2)
After cutting and crushing 1 kg of dry weight of coconut (Lycium chinense), 10 L of Milli-Q water was added, and autoclaving was performed at 105 ° C. for 15 minutes. Centrifugation was performed at 4 ° C. and 3000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter to obtain an extract. The obtained liquid was concentrated and dried to obtain about 87 g of a solid.

(Example 3) β-hexosaminidase release assay In a medium prepared with 10% FBS, MEM 4.7 g / 500 ml, 200 mM L-glutamine 5 ml / 500 ml, 10% NaHCO ₃ (pH 7.0 to 7.4) Human juvenile basophil (KU812) cells are seeded and cultured at 37 ° C. under 5% CO ₂ . Next, the cultured KU812 is peeled off from the flask, prepared using the same medium as described above to a cell concentration of 5 × 10 ⁵ cells / ml, and finally 1 mg / ml mouse anti-DNP-IgE antibody is added at 0.3 ml / ml. Add to ml. After suspending well, the cells were seeded at 100 μl / well in a 96-well plate and incubated overnight at 37 ° C. under 5% CO ₂ to sensitize the cells. The next day, after washing the cells twice with 200 μl PBS, β-hexosaminidase release buffer (116.9 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO ₄ .7H ₂ O, 5.6 mM glucose, 25 mM HEPES, 2.0 mM CaCl ₂ , 1.0 mg / ml BSA dissolved in 500 ml of water, adjusted to pH 7.7 with 10% NaHCO ₃ and filter sterilized) 60 μl and wolfberry extract diluted sample prepared in Example 1 ( 1 / 100,1 / 1000, / 10000 dilution) or adding 5μl control (PBS), under 5% CO _2, incubated 10 min at a carbon dioxide gas incubator at 37 ° C.. Next, 5 μl of 4 μg / ml DNP-BSA was added and incubated in a carbon dioxide incubator at 37 ° C. under 5% CO _{2 for} 1 hour, and then the substrate solution (85.5 mg of p-nitrophenyl-N was added to 20 μl of the supernatant. Add 80 μl of -acetyl bD-glucosamide in 50 ml of 50 mM citrate buffer (pH 4.5; pH adjusted with citric acid) and incubate at 37 ° C. for 30 minutes. Thereafter, 200 μl of a reaction stop solution (100 mM carbonate buffer; Na ₂ CO 3 1.06 g / 100 ml, NaHCO 3 0.84 g / 100 ml) is added, and the absorbance (A ₄₅₀ ) at 450 nm is measured with a microplate reader. At this time, the inhibition rate of β-hexosaminidase release is calculated by the following formula.

Inhibition rate (%) = {1− (SB) / (C−b)} × 100
S: A ₄₅₀ when sample is added to cells
B: A ₄₅₀ when sample was added in the absence of cells
C: A _{450 of} control (when PBS is added to the cells)
b: A ₄₅₀ when control is added in the absence of cells
The results are shown in FIG.

  The wolfberry extract of the present invention significantly inhibited β-hexosaminidase release from basophils, which is an indicator of antiallergic action.

(Example 4) Manufacture of wolfberry extract-containing tablets 45 parts by weight Lactose obtained in Example 2 35 parts by weight Lactose 35 parts by weight Crystalline cellulose 15 parts by weight Sucrose fatty acid ester 5 parts by weight Eating and drinking tablets containing wolfberry extract were produced.

(Example 5) Production of wolfberry extract-containing soft capsules wolfberry extract obtained in Example 2 40 parts by weight olive oil 55 parts by weight glycerin fatty acid ester 5 parts by weight The above composition was mixed, pulverized, and filled into gelatin capsules. A food and beverage capsule containing a wolfberry extract was produced.

(Example 6) Production of soft drink containing wolfberry extract wolfberry extract obtained in Example 2 1 part by weight liquid sugar 30 parts by weight citric acid 0.1 part by weight perfume 0.5 part by weight water 150 parts by weight The above composition To produce a soft drink containing wolfberry extract.

(Example 7) Preparation of wolfberry extract-containing cracker wolfberry extract obtained in Example 2 1 part by weight weak flour 120 parts by weight salt 1 part by weight baking powder 2 parts by weight butter 30 parts by weight water 40 parts by weight A wolfberry extract-containing cracker was prepared by

(Example 8) Preparation of dressing containing wolfberry extract wolfberry extract obtained in Example 2 10 parts by weight olive oil 80 parts by weight vinegar 60 parts by weight salt 3 parts by weight pepper 1 part by weight lemon water 5 parts by weight A dressing containing wolfberry extract was prepared.

(Example 9) Preparation of chewing gum containing wolfberry extract wolfberry extract obtained in Example 2 0.5 parts by weight gum base 20 parts by weight sugar 70 parts by weight perfume 0.5 parts by weight water A product-containing chewing gum was prepared.

(Example 10) Preparation of tea containing wolfberry extract An appropriate amount of wolfberry extract obtained in Example 2 is dissolved in hot water.

Claims (20)

Antiallergic agent containing Lycium plant or polar solvent extract as an active ingredient. A basophil cell degranulation inhibitor comprising a Lycium plant or a polar solvent extract thereof. A mast cell degranulation inhibitor comprising a Lycium plant or a polar solvent extract thereof. A β-hexosaminidase release inhibitor comprising a Lycium plant or a polar solvent extract thereof. An antiallergic agent comprising one or more of the degranulation inhibitor according to claims 2 to 4 and the β-hexosaminidase release inhibitor. The antiallergic agent according to claim 1 or 5, wherein the plant of the genus Lycium is Lycium chinense. The licorice (Lycium) extract is an extract obtained by extracting wolfberry with water or a mixed solvent of water and a polar organic solvent, according to any one of claims 1, 5 and 6. Antiallergic agent. A food or drink comprising the antiallergic agent according to any one of claims 1, 5, 6 and 7. A cosmetic comprising the antiallergic agent according to any one of claims 1, 5, 6 and 7. A bath preparation comprising the antiallergic agent according to any one of claims 1, 5, 6 and 7. A pharmaceutical comprising the antiallergic agent according to any one of claims 1, 5, 6 and 7. The allergy prevention or treatment method using the antiallergic agent of any one of Claim 1, 5, 6 or 7. An anti-inflammatory agent comprising any one or more of the degranulation inhibitor and the β-hexosaminidase release inhibitor according to claim 2. The anti-inflammatory agent according to claim 13, wherein the plant of the genus Lycium is Lycium chinense. The anti-inflammatory according to any one of claims 13 and 14, wherein the Lycium extract is an extract obtained by extracting wolfberry with water or a mixed solvent of water and a polar organic solvent. Agent. The food / beverage products characterized by mix | blending the anti-inflammatory agent of any one of Claims 13-15. A cosmetic comprising the anti-inflammatory agent according to any one of claims 13 to 15. A bath preparation comprising the anti-inflammatory agent according to any one of claims 13 to 15. A pharmaceutical comprising the anti-inflammatory agent according to any one of claims 13 to 15. The inflammation prevention or treatment method using the anti-inflammatory agent of any one of Claims 13-15.

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