JP5403320B2 - Degreasing inhibitor, β-hexosaminidase release inhibitor, antiallergic agent and anti-inflammatory agent obtained from natural extract - Google Patents

Description

  The present invention relates to a degranulation inhibitor or β-hexosaminidase release inhibitor that has a degranulation inhibitory effect or a β-hexosaminidase release inhibitory effect and is effective for various inflammations of allergic diseases such as hay fever. More specifically, a degranulation inhibitor that exhibits an anti-inflammatory effect in a small amount, has no side effects such as sedation and hypnosis, is not impaired in flavor even when added to food, and can be taken continuously over a long period of time. Or β-hexosaminidase release inhibitors and their novel uses.

  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. It is divided into delayed allergy.

  Basophils present in blood, and mast cells present in the vicinity of blood vessels and connective tissues have granules containing chemical mediators such as histamine, thromboxane A2, and leukotriene. -It is known to liberate enzymes such as hexosaminidase 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. .

  Substances released by degranulation of basophils and mast cells (chemical mediators) are also 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 suppressing degranulation of these cells. Therefore, it is desired to develop an effective basophil cell degranulation inhibitor or mast cell degranulation inhibitor and provide a useful antiallergic agent.

  Anti-allergic agents include drugs that suppress degranulation from mast cells and chemical mediator release 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, but there are side effects such as drowsiness and sedation, central nervous system inhibitory effects, hematuria and other cystitis-like symptoms, and their use It requires strict guidance by a doctor, and the user is required to be careful not to take it when driving.

  In recent years, research on Kampo medicine using herbal medicine has attracted attention. Kampo therapy is a treatment using natural medicines derived from natural products, and although it is slightly delayed, it has the advantage that the medicinal effect is systemic because it can improve not only the local symptoms but also the constitution. is there. Many drugs have fewer side effects than drugs and are safe even if taken for a long time. As Kampo medicines used for the above-mentioned Kampo therapy, Kakkonto, Shoseiryu, Koshigaya-Koryu, etc. are known. These herbal medicines are composed of several kinds of herbal medicines. For example, in the case of Kakkonto, herbal medicines such as kakkon, mah, cinnamon, glaze, daikon, licorice and ginger are included.

  These herbal medicines are used in the form of granules, tablets and the like, but often have problems of flavor such as peculiar bitterness and poor aftertaste, and have a drawback of being difficult to take. Therefore, it may be easier to take the constituent herbal ingredients in Chinese herbal medicines in the form of processed foods, foods such as beverages, but some of these herbal medicines are not approved as food. There are also things. Moreover, even if it can be used for food, it has a peculiar odor and taste, and therefore, it often impairs the original flavor of food. Also, commercially available antiallergic agents and foods do not exert effects on inflammation in general and are often symptomatic treatments. Therefore, when symptoms such as hay fever are combined, they often do not exhibit sufficient effects.

  Moreover, even when used for improving skin inflammation as a cosmetic, many Chinese herbal medicines cause problems such as browning of products and sensitization.

  By the way, guarana (scientific name: Paulinia cupana Kunth.) Is a plant belonging to the family Muclidaceae, and its seeds contain caffeine and other caffeine-related substances such as galanin and xanthines. Have been used as beverages. Since caffeine and its related substances contained in guarana are metabolized more slowly than caffeine alone, it is said that it has a mild and sustained action, is not addictive, and is safe if taken properly (Non-Patent Documents) 1, Non-Patent Document 2). In recent years, it has been widely used for cola drinks, chocolates and the like.

  As the pharmacological effects of guarana, nerve tonicity, migraine improvement, obesity improvement, blood pressure increase at low blood pressure, etc. have been reported. In addition to nerve tonics, excitable drinks (athlete dietary supplements, etc.), various It is also used for health medicine.

  However, there have been no reports so far regarding galana's degranulation inhibitory action, β-hexosaminidase release inhibitory action, antiallergic action, and anti-inflammatory action.

National Institute of Health and Nutrition, “Safety and Efficacy Information for“ Health Foods ”” [Search September 9, 2008], Internet <http://hfnet.nih.go.jp/contents/ detail498.html> Toshio Shimizu et al., “Functional Food Material Handbook”, Yakuji Nipposha (2004), p. 273-274

  The object of the present invention has been made in view of such circumstances, and is expected to have high antiallergic action or anti-inflammatory action, has no side effects such as sedation / hypnosis, and has high safety, and is also a raw material Basophil cell degranulation inhibitor, mast cell degranulation inhibitor or β-hexosaminidase release inhibitor, and anti-allergic or anti-inflammatory foods that are easy to obtain and manufacture and that have a mild flavor Another object of the present invention is to provide pharmaceuticals, animal feed compositions, and cosmetic raw material compositions.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have obtained a polar solvent extract obtained by extracting from a fruit or seed of guarana (Paullinia cupana Kunth.) Using a polar solvent ( (Hereinafter simply referred to as “extract”) has a basophil cell degranulation inhibitory action, a mast cell degranulation inhibitory action and a β-hexosaminidase release inhibitory action, and has an excellent antiallergic action or antiinflammatory action. As a result, the present invention has been completed.

  That is, the basophil cell degranulation inhibitor of the present invention is characterized by comprising an extract of a fruit or seed of a guarana (Paullinia cupana Kunth.) Plant as an active ingredient.

  The mast cell degranulation inhibitor of the present invention is characterized by containing an extract of fruit or seed of a guarana (Paullinia cupana Kunth.) Plant as an active ingredient.

  In addition, the β-hexosaminidase release inhibitor of the present invention is characterized by containing a fruit or seed extract of a guarana (Paullinia cupana Kunth.) Plant as an active ingredient.

  The food, medicine, animal feed composition and cosmetic raw material composition having an antiallergic action or antiinflammatory action according to the present invention are the basophil cell degranulation inhibitor, the mast cell degranulation inhibitor and the β-hex. It contains any one or more of sosaminidase release inhibitors.

  In addition, the basophil cell degranulation inhibitor, mast cell degranulation inhibitor and β-hexosaminidase release inhibitor of the present invention may be any preventive or ameliorating agent for allergic action or inflammatory action. Of course, both of them may be used.

  The extract of the fruit or seed of the guarana plant in the present invention (hereinafter sometimes referred to as “guarana extract”) is useful as an antiallergic agent or an antiinflammatory agent. The guarana extract of the present invention has no side effects such as sedation / hypnosis that are commonly found in existing antiallergic agents and anti-inflammatory agents, but rather stimulates the central nerve and has a wakefulness / nerve tonicity. It can be used without worrying about the action and the like. In addition, the guarana extract of the present invention can be prepared by a simple method and has a mild flavor, so it is highly applicable to pharmaceuticals, foods and drinks, cosmetics, bath preparations, animal feeds, etc. Is expected to be very wide.

Hereinafter, an embodiment of the present invention will be described in detail.
The guarana extract in the present invention is produced using fruits or seeds of guarana (Paullinia cupana Kunth.) Plants as raw materials.

  The production method of the guarana plant used in the present invention is not particularly limited, and it may be not only a self-generated one but also an artificially cultivated one. Moreover, it does not restrict | limit especially about the collection time of the guarana plant used by this invention, the growing years, the cultivation method, and the cultivation period.

  The method for producing the guarana extract is not particularly limited, and can be produced by a commonly used method. The extraction conditions are not particularly limited, and for example, the seeds and / or fruits are produced by being extracted with a polar solvent such as water or alcohol after being cut as it is or after being cut, pulverized or finely ground.

  Extraction using a polar solvent may be carried out for about 10 minutes to 1 week under normal temperature to increased pressure under normal temperature to the boiling point of the solvent according to the solvent used. The polar solvent used for the extraction may be appropriately selected and used according to the plant species and the treatment process. For example, in addition to water, alcohols (for example, lower alcohols such as methanol, absolute ethanol and ethanol, or propylene glycol) Organic solvents such as polyhydric alcohols such as 1,3-butylene glycol), ketones such as acetone, organic acids such as acetic acid, and the like. These solvents can be used alone, but two or more kinds can be used in any combination. In addition, when the residue of an organic solvent is not preferable like the case where it uses as food, it is preferable to use water, ethanol, hydrous ethanol, etc. especially. The extraction method is not particularly limited, and normal temperature homogenization extraction, far infrared extraction, ultrasonic irradiation extraction, reflux extraction, supercritical fluid extraction, and the like can be used.

  Specifically, for example, the following method can be used. That is, after harvesting guarana seeds and fruits as they are, or by pulverizing the dried and matured fruits, add 5 to 20 times the amount of extraction solvent, leave at normal pressure at room temperature for about one week, or near the boiling point of the extraction solvent The filtrate obtained by extraction for about 10 to 30 minutes and filtered is used as a liquid as it is, or the filtrate is dried under reduced pressure or freeze-dried to obtain a guarana extract.

  The guarana extract obtained as described above can be used as it is, but if necessary, further purification treatment such as deodorization and decolorization may be added within a range not affecting the efficacy. Such a purification treatment may be carried out by arbitrarily selecting ordinary means. For example, filtration, ion exchange resin, activated carbon column, etc. may be used for adsorption, decolorization, purification, etc. Furthermore, a purified product in the form of a solution, paste, gel, or powder can be obtained by freeze drying, concentration treatment, or the like.

  Further, in order to improve the properties of the guarana extract, it may be appropriately modified using an enzyme such as tannase, glucosidase, laccase, peroxidase, cellulase, pectinase.

  The form of the guarana extract in the present invention is not particularly limited, and the extract obtained by production may be used as it is, but for example, it consists of the extract and other components added as necessary. It may be used in the form of a composition. Examples of such a composition include a composition comprising the above extract and an appropriate carrier (such as food or medicine, dextrin used in cosmetics). Specific examples of these compositions include forms such as foods (food and drinks) suitable for ingestion, pharmaceuticals, cosmetic raw materials, animal feeds, additives for animal feeds, and the like. When the guarana extract is ingested in such a form, the dose of the extract to a human or mammal is usually in the range of 0.01 to 2000 mg / kg body weight per day, desirably Although it is the range of 0.1-200 mg / kg body weight per day, it is not limited to this range.

  For use in food or animal feed, the above extract is mixed with other various components, for example, solid food or animal feed, creamy or jammed semi-fluid food or animal feed, gel food or animal feed. Prepare in the form of beverages. In addition, the extract may be blended as it is in food or animal feed, or it may be blended in the form of powder, granules, capsules, tablets, liquids, etc. produced by combining with an appropriate carrier as necessary. May be.

  There are no particular limitations on the other components that are blended in the food or animal feed together with the above extract, and any of various commonly used components can be used. Examples of such components include glucose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, glycerin, propylene glycol, Glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B group, nicotinamide, calcium pantothenate, amino acids , Calcium salts, pigments, fragrances, preservatives and the like, and these may be appropriately blended depending on the type of food or animal feed.

  Specific examples of the food include soft drink, juice, coffee, tea, liqueur, milk, whey drink, lactic acid bacteria drink, candy, chewing gum, chocolate, gummi, yogurt, ice cream, pudding and the like. Specific examples of animal feed include dog food, cat food, livestock mixed feed, and the like. The content of guarana extract in food or animal feed is suitably in the range of 0.01 to 100 mg / g, but is not limited to this range.

  For use in the form of a pharmaceutical preparation, a usual pharmaceutically acceptable carrier is added to the above extract to prepare it in a solid, semi-solid or liquid form. Specific examples include tablets, capsules, pills, granules, powders, emulsions, suspensions, syrups, pellets and other oral administration agents, ointments and other external preparations, injections and the like. Examples include parenteral administration agents.

  The dosage of the above pharmaceutical preparation may be appropriately adjusted according to the dosage form, disease, etc., but if it is an internal preparation, the guarana extract in the total amount of the preparation is 0.5 to 99 mass% in terms of solid content Preferably, it may be blended in the range of 5 to 90% by mass. As an external preparation, the content of guarana extract in a pharmaceutical preparation is suitably in the range of 0.01 to 50 mg / g, but is not limited to this range.

  Examples of cosmetics include lotions, emulsions, lotions, tonics, cosmetic creams, sprays, lipsticks, foundations, shampoos, rinses, bathing agents and the like. These cosmetics are produced by mixing guarana extract with a commonly used cosmetic base. Examples of external preparations include ointments, creams, liquids, patches, and the like, which can be produced in combination with conventional pharmaceutical carriers. The content of guarana extract in these cosmetics and external preparations is suitably in the range of 0.001 to 100 mg / g, but is not limited to this range.

  When formulating, carriers such as surfactants, excipients, binders, disintegrants, lubricants, preservatives, stabilizers, buffers, suspensions and the like that have been conventionally used depending on the dosage form are used. Can be used. Preferably, for example, solid carriers such as starch, lactose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, distilled water, physiological saline, glucose aqueous solution, alcohols such as ethanol, liquid carriers such as propylene glycol and polyethylene glycol, and various types And oily carriers such as animal and vegetable oils, white petrolatum, paraffin and wax.

  These guarana extract-containing foods, pharmaceuticals, animal feed compositions and cosmetic raw material compositions have basophil cell degranulation inhibitory action, mast cell degranulation inhibitory action and β-hexosaminidase release inhibitory action. Thereby having anti-allergic and anti-inflammatory effects. Therefore, it is effective for prevention, improvement and treatment of various disorders and diseases caused by chemical mediators released by basophil cells and mast cells present in the living body.

  In the present invention, the basophil cell degranulation inhibitory action or mast cell degranulation inhibitory action is evaluated by measuring the enzyme activity of β-hexosaminidase released from the cells by degranulation.

  β-hexosaminidase is one of the chemical mediators released by leukocytes, and is known to be released almost simultaneously with histamine, which is a causative agent of allergies. Rat leukemia cells (RBL-2H3) are known to have the same functions as human basophils and mast cells, and β-hexosaminidase release inhibitory effect from RBL-2H3 is an inhibitor of type I allergy. It is considered as an indicator of effectiveness. Therefore, the anti-inflammatory effect can be evaluated by measuring β-hexosaminidase release inhibition from RBL-2H3. Conventionally, hyaluronidase inhibitory activity and 3-α-HSD inhibitory activity are known as indicators of anti-inflammatory effects, but depending on inflammation, this indicator alone may not be sufficient to judge, particularly for immediate allergies. In some cases, these indicators alone cannot be used for judgment. In addition, evaluating the β-hexosaminidase release inhibitory effect has a higher correlation with in vivo evaluation than other evaluation methods.

  The present invention will be described more specifically with reference to production examples and examples. However, the scope of the present invention is not limited to these examples.

<Method for preparing guarana extract>
100 g of dried guarana (Paullinia cupana Kunth.) Seeds as raw materials were added to 0.9 L of 50% ethanol (ethanol: water = 50: 50 (weight ratio)), refluxed for 2 hours, and cooled to room temperature. Filtered and the filtrate was left refrigerated overnight. Subsequently, the filtrate was further filtered, and the obtained filtrate was concentrated under reduced pressure and further freeze-dried to obtain a guarana extract.

細胞内全β−ヘキソサミニダーゼの測定には、2 ％ Triton X-100を加えて溶解した細胞を遠心分離した上清を用いた。
ガラナ抽出物等のサンプル添加によるβ−ヘキソサミニダーゼ遊離阻害率は以下の式によって求めた。

さらに、細胞のβ−ヘキソサミニダーゼ遊離を50%抑制するのに必要なサンプル量（IC₅₀）を求め、IC₅₀にて活性の強さを評価した。 Test Example 1 <β-Hexosaminidase Release Test>
Rat leukemia cells (RBL-2H3) were seeded in a 24-well plate at 9.6 × 10 ⁴ cells / well (400 μl well) and cultured at 37 ° C. for 24 hours. After culture, RBL-2H3 cells were washed with Tyrode buffer (+) (137 mM NaCl, 5.6 mM Glucose, 11.9 mM NaHCO ₃ , 2.7 mM KCl, 41.7 mM NaH ₂ PO ₄ , 1 mM CaCl ₂ , 0.5 mM MgCl ₂ ) did. Calcium ionophore A23187 (SIGMA, final concentration 10 μM) that promotes degranulation was added and reacted at 37 ° C. for 30 minutes. Each sample solution was added before A23187 was added. Each sample solution was prepared by adding each extract shown in Table 1 containing guarana extract and a predetermined amount of caffeine to 30% ethanol.
The 24-well plate was centrifuged for 3 minutes at 1500 rpm, and 100 μl of each supernatant was placed in a 96-well plate. After adding 1 mM p-nitrophenyl-N-acetyl β-D-glucosaminide (PNAG) and mixing, react in a CO ₂ incubator at 37 ° C for 90 minutes, and add 100 μl stop buffer (0.2 M glycine, pH) to the reaction solution. 11.0) was added. Absorbance was measured with a microplate reader Model 550 (BIO-RAD), and the release rate of 竈 -hexosaminidase was determined by the following formula (measurement wavelength 405 nm, reference wavelength 610 nm). The absorbance of only the test substance was also measured and corrected. Three wells were measured for each, and the average value was determined.

For measurement of intracellular total β-hexosaminidase, a supernatant obtained by centrifuging cells dissolved by adding 2% Triton X-100 was used.
The inhibition rate of β-hexosaminidase release by adding a sample such as guarana extract was determined by the following equation.

Furthermore, the amount of sample (IC ₅₀ ) required to suppress the release of β-hexosaminidase from cells by 50% was determined, and the activity intensity was evaluated by IC ₅₀ .

In addition, as a positive control, β-hexosaminidase release from extracts of karin, wolfberry leaves, summer savory, peach leaves, wormwood, rose hips and rosemary, which have already been reported to have antiallergic or anti-inflammatory effects. The inhibitory effect was measured and compared with guarana extract. In addition, the extract of these samples was extracted by the same operation as the guarana extract using 30-50% ethanol as a solvent. IC ₅₀ was also measured in the same manner for caffeine, one of the main components of guarana. The results are shown in Table 1.

IC ₅₀ represents the amount of sample required to inhibit the release of β-hexosaminidase from cells by 50%, and the lower the value, the stronger the activity. As a result of the measurement, the guarana extract of the present invention suppresses the release of β-hexosaminidase of cells, which is an index of antiallergic action and anti-inflammatory action, and more than the previously reported karin extract and rosemary extract. It was strong.

  Guarana is rich in caffeine. It is described that caffeine has an antiallergic action (Tetsuya Shiozaki et al., “Effects of tea extract, catechin, and caffeine against type I allergy”, Pharmaceutical Journal (1997) 117, p448-457). However, when caffeine's β-hexosaminidase release inhibitory action was examined, as shown in Table 1, no β-hexosaminidase release inhibitory action was observed, and thus basophil cell degranulation inhibitory action and obesity were observed. Since it does not have a cell degranulation inhibitory effect, it is presumed that the β-hexosaminidase release inhibitory effect of guarana extract is caused by components other than caffeine.

Test Example 2 <Mouse IgE-stimulated β-hexosaminidase release test>
Rat leukemia cells (RBL-2H3) were seeded in a 24-well plate at 4.8 × 10 ⁴ cells / well (400 μl / well) and cultured at 37 ° C. for 24 hours. After the culture, an anti-dinitrophenyl IgE antibody (SIGMA) was added to the culture solution so that the final concentration was 1 μg / ml. The cells were further cultured at 37 ° C. for 1 hour, and RBL-2H3 cells were washed with Tyrode buffer. A sample solution containing a guarana extract was added, and then DNP-HSA (SIGMA) was added to a final concentration of 1 μg / ml. The cells were cultured at 37 ° C for 2 hours. Subsequently, using the same method as in Test Example 1, the β-hexosaminidase release rate of the culture supernatant and the IC _{50 of the} guarana extract were measured.

As a result, IC ₅₀ of guarana extract was 16.0 μg / ml. The guarana extract also suppressed the release of β-hexosaminidase in cells induced by human IgE stimulation.

Test Example 3 <Measurement of Live Cell Number of RBL-2H3 Cells>
Rat leukemia cells (RBL-2H3) were seeded in a 24-well plate at 3.6 × 10 ⁵ cells / well (500 μl well). A sample solution containing guarana extract (final concentration: 200, 100, 50, 0 μg / ml) was added and cultured in a CO ₂ incubator for 24 hours. After the cells were collected, they were suspended in PBS and suspended at a ratio of 1: 1 with 0.5% trypan blue (Wako Pure Chemical Industries). The viable cell count was counted with a hemocytometer.
The ratio (%) of the number of viable cells at each sample concentration relative to the control was calculated by the following formula. The results are shown in Table 2 as the survival rate.

  As shown in Table 2, the guarana extract had no effect on the survival of RBL-2H3 cells at 100 μg / ml, and the survival rate was almost 90% even at a high dose of 200 μg / ml. From this experiment, it can be seen that the guarana extract did not inhibit the degranulation by suppressing the proliferation of RBL-2H3 cells, that is, the guarana extract suppressed the degranulation without killing the RBL-2H3 cells. Moreover, even if it coexists for 24 hours, there is little damage to a cell and it is thought that there is little cytotoxicity.

Test Example 4 <Measurement of Viable Cell Number of RBL-2H3 Cells>
Furthermore, in order to examine the influence of the guarana extract on the growth of RBL-2H3 cells, the number of viable cells was measured when the guarana extract concentration was 400 μg / ml by the same method as in Test Example 3. However, the culture time of the cells after adding the guarana extract was set to 30 minutes as in Test Example 1.

  As a result of the measurement, the viability of RBL-2H3 cells was 97% when exposed to a guarana extract concentration of 400 μg / ml for 30 minutes, and the guarana extract had little effect on the survival of RBL-2H3 cells. . On the other hand, as shown in Table 1, the guarana extract strongly suppressed β-hexosaminidase release in RBL-2H3 cells after 30 minutes of culture. This experiment also shows that the guarana extract did not suppress the degranulation by suppressing the growth of RBL-2H3 cells.

Claims (5)

A basophil cell degranulation inhibitor comprising a water-containing ethanol extract of fruit or seed of a guarana (Paullinia cupana Kunth.) Plant as an active ingredient. A mast cell degranulation inhibitor comprising a hydrous ethanol extract of fruit or seed of a guarana (Paullinia cupana Kunth.) Plant as an active ingredient. Β-hexosaminidase release inhibitor containing a water-containing ethanol extract of fruits or seeds of guarana (Paullinia cupana Kunth.) Plants as an active ingredient. Antiallergic agent containing water-containing ethanol extract of fruit or seed of guarana (Paullinia cupana Kunth.) Plant as an active ingredient . Anti-inflammatory agent containing hydrous ethanol extract of fruit or seed of guarana (Paullinia cupana Kunth.) Plant as an active ingredient.