JP5783404B2 - Hyaluronidase inhibitor - Google Patents

Description

  The present invention relates to a hyaluronidase inhibitor containing psyllium seed gum as an active ingredient.

  Hyaluronic acid is a kind of mucopolysaccharide that is abundant in tissues such as skin and joint fluid.For example, in the skin, cell protection, nutrient transport, tissue moisture retention, flexibility maintenance, etc. As synovial fluid, it plays an important role in maintaining the tissue structure / function and maintaining lubricity.

  The amount of hyaluronic acid in living bodies such as skin and joints decreases due to aging or pathological conditions, which may cause dry skin, decreased elasticity, increased wrinkles, rough skin, joint pain due to deterioration of joint wettability, etc. cause. On the other hand, by inhibiting hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, it is possible to suppress the degradation of hyaluronic acid and contribute to the maintenance of the amount of biological hyaluronic acid.

  On the other hand, hyaluronidase inhibitors can also be used as anti-inflammatory / anti-allergic agents caused by hyaluronidase. This is because hyaluronidase is activated during inflammation, thereby destroying the matrix of connective tissue and enhancing the infiltration of inflammatory tissues and the permeability of blood vessels. In addition, hyaluronidase is likely to mediate in the process of histamine release from mast cells in type I allergy.

  Therefore, hyaluronidase inhibitors are increasing in number of patients in recent years, and are expected to prevent or improve allergic diseases such as atopic dermatitis, hay fever, bronchial asthma and food allergies, which are becoming social problems. For example, anti-allergic sodium cromoglycate and tranilast, and anti-inflammatory drugs aspirin and indomethacin have hyaluronidase activity inhibitory activity, suggesting that this inhibitory role plays a role in exerting therapeutic effects. (Non-patent Documents 1 and 2).

  So far, compounds having hyaluronidase inhibitory activity extracted from natural products include tea polyphenols extracted from tea used for drinking (Patent Document 1), chimneys used as crude drugs or foods and drinks. Extracts of pheasants and rahan fruit (Patent Document 2), extract of urajirogashi which is a beech family plant (Patent Document 3), cashew nut shell oil (Patent Document 4), plant extract of Urushiaceae (Patent Document 5), seaweed ( Patent Literature 6) and the like have been found, but safety is not sufficiently confirmed, action / effect is insufficient, and it is difficult to stably add to the preparation while maintaining activity. Such problems remain.

JP-A-6-99391 JP-A-6-80576 JP-A-6-239757 JP-A-6-329526 Japanese Patent Laid-Open No. 7-10765 JP-A-9-67266

Chem. Pharm. Bull. 33, 642 (1985) Kakegawa Toshio et al., Inflammation, 4,437 (1984)

  Therefore, the main object of the present invention is to provide a hyaluronidase inhibitor having excellent hyaluronidase inhibitory activity and excellent safety.

  As a result of intensive studies to achieve the above object, the present inventor has found that a low-molecular psyllium seed gum exhibits excellent hyaluronidase inhibitory activity, and has completed the present invention.

  That is, this invention relates to the hyaluronidase inhibitor which uses low molecular weight psyllium seed gum as an active ingredient.

  According to the present invention, a highly safe and highly active hyaluronidase inhibitor can be provided.

It is a chromatogram which shows the GPC analysis result of a low molecular weight psyllium seed gum (A) and unfractionated (B) psyllium seed gum. FIG. 6 is a graph showing the IC ₅₀ of small molecules and unfractionated psyllium seed gum.

(1) Psyllium seed gum Psyllium seed gum is water-soluble dietary fiber purified from the skin of the plant seeds of the plantain family. In the present invention, a commercially available psyllium seed gum can be used, and for example, a psyllium seed gum extracted from the seed coat of Plantago ovata can be used.

  The extraction method of the psyllium seed gum is not limited, and for example, a known method such as alcohol dehydration can be used. Alcohol dehydration is a method of dissolving polysaccharides (silium seed gum) -containing raw materials in hot water, filtering off insoluble substances such as proteins and fibers, and adding a hydrophilic organic solvent to the filtrate to precipitate polysaccharides. The precipitate is dried and pulverized.

  In addition, a method for purifying water-soluble polysaccharide (silium seed gum) raw material powder by decomposing insoluble substances such as proteins and fibers with water or a hydrophilic organic solvent using an enzyme (JP-A-60-118152). No. 1-5041, JP-A-1-266101, JP-A-1-193302); water-soluble polysaccharide powder and insoluble substances such as proteins and fibers in an organic solvent or air Separation and purification using specific gravity difference (JP-B-42-14034, JP-B-60-17201, etc.); water-soluble polysaccharide powder and insoluble substances such as protein and fiber May be used by separating and purifying the solution according to the difference in behavior when moving under the influence of gravity and centrifugal force in an aqueous solution of a hydrophilic organic solvent (Japanese Patent Publication No. 2-40682). it can.

(2) Low molecular weight psyllium seed gum The low molecular weight psyllium seed gum used in the present invention has a weight average molecular weight of 1000 to 100,000, preferably 1000 to 50000, as measured by gel permeation chromatography. The low-molecular psyllium seed gum used in the present invention is a B-type viscometer (manufactured by Tokyo Keiki Co., Ltd.) at 25 ° C., 30 rpm, rotor No. The viscosity when measured under the condition 1 is 200 mPa · s or less, preferably 100 mPa · s or less.

  The low molecular psyllium seed gum of the present invention can be a commercially available product (for example, manufactured by MRC Polysaccharide). It can also be obtained by a known method. For example, the refined psyllium seed gum described in (1) may be hydrolyzed with acid, alkali, heat, enzyme, etc. to reduce the molecular weight (decrease the molecular weight) or reduce the viscosity. it can. The hydrolysis conditions are not limited as long as a desired low-molecular psyllium seed gum is obtained, and can be selected as appropriate. For example, low molecular psyllium seed gum can be obtained by adding 2N hydrochloric acid (0.5 mL) to 1% psyllium seed gum aqueous solution (200 mL) and treating at 80 ° C. for 30 minutes.

  The method for purifying (fractionating) a commercially available product or the low molecular psyllium seed gum thus obtained by hydrolysis is not limited, and can be carried out by a known method. For example, a known method such as an ultrafiltration membrane method can be used. The ultrafiltration membrane method is a separation method using a membrane for a high molecular weight substance having a molecular weight of about 10,000 to 3,000,000. The ultrafiltration membrane is an asymmetric membrane composed of a skin layer and a sponge layer, and allows water, ionic molecules, and low molecular weight materials to permeate without permeating high molecular weight materials. Moreover, the low molecular weight psyllium seed gum used in the present invention can also be prepared by molecular weight fractionation using gel filtration chromatography.

(3) Hyaluronidase Inhibitor The present inventor has found for the first time that the low molecular weight psyllium seed gum has a high hyaluronidase inhibitory activity. Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid, and the subtype or the like whose activity is inhibited in the present invention is not limited.

  The measuring method of hyaluronidase activity is not limited and a known method can be used. For example, a method using the Morgan-Elson method (J. Biol. Chem. 1955, 217: 959-966) is preferable.

  The hyaluronidase inhibitor of the present invention can be used, for example, at 0.01 to 100 mg / mL, preferably 0.1 to 10 mg / mL with respect to 1 mg / mL of hyaluronidase.

  The low molecular weight psyllium seed gum of the present invention can be used as an antiallergic agent. The type of allergy to be targeted is not limited, and examples include atopic dermatitis, hay fever, bronchial asthma, food allergy, and the like. The low molecular psyllium seed gum of the present invention can also be used as an anti-inflammatory agent.

(4) Pharmaceuticals The hyaluronidase inhibitor of the present invention can be used as pharmaceuticals (including pharmaceuticals and quasi drugs). The above pharmaceutical products can be used for humans or for animals other than humans.

  The hyaluronidase inhibitor of the present invention can be appropriately selected depending on the dosage form. It can be produced by appropriately blending. Examples of additives that can be incorporated into the hyaluronidase inhibitor of the present invention include excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cocoa butter, hydrogenated vegetable oil, kaolin, talc, and the like. ), Binder (distilled water, physiological saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrant (agar, sodium bicarbonate, calcium carbonate) , Sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, gelatin, ethanol, etc.), disintegration inhibitors (sucrose, stearin, cocoa butter, hydrogenated oil, etc.), absorption enhancers (quaternary ammonium base, sodium lauryl sulfate, etc.) ), Adsorbent (glycerin, starch Lactose, kaolin, bentonite, silicic acid, etc.), lubricants (purified talc, stearates, polyethylene glycol, and the like) and the like.

  The administration method of the hyaluronidase inhibitor according to the present invention can be generally administered orally in the form of tablets, pills, soft / hard capsules, fine granules, powders, granules and the like. The water-soluble preparation can be administered orally as a liquid. Furthermore, parenteral administration may be used. When administered as a parenteral agent, the hyaluronidase inhibitor of the present invention is dispersed in a suitable solubilizing agent such as ethanol or water, and then in a dosage form such as a poultice, lotion, ointment, tincture or cream. Can be applied. The water-soluble preparation of this hyaluronidase inhibitor can be used as it is or in the form of a poultice, lotion, ointment, tincture, cream, etc. with a dispersant, suspension, stabilizer, etc. added. Can be applied.

  The compounding ratio when using the hyaluronidase inhibitor of the present invention as a drug can be appropriately changed depending on the dosage form, but is usually about 0.01 to 10 wt% when administered orally or by mucosal absorption, In the case of parenteral administration, it should be about 0.01 to 20 wt%. In addition, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or it may be necessary to administer beyond the range. In addition to the hyaluronidase inhibitor, the pharmaceutical composition may include other compounds commonly used in the pharmaceutical field and compounds necessary for molding into a form suitable for oral administration. Examples of such compounds include lactose, starch, hydroxypropyl cellulose, kaolin, talc, calcium carbonate and the like.

  Even if the hyaluronidase inhibitor of the present invention is used as an external preparation for skin (including cosmetics, pharmaceuticals and quasi drugs), it can be expected to inhibit hyaluronidase. In addition, the said skin external preparation may be used for a human and may be used for mammals other than a human. Examples of the external preparation for skin to which the hyaluronidase inhibitor of the present invention can be formulated include, for example, emulsion, soap, facial cleanser, bath preparation, cream, emulsion, lotion, eau de cologne, shaving cream, shaving lotion, makeup Oil, sunscreen / sunscreen lotion, funny powder, foundation, perfume, pack, nail cream, enamel, enamel remover, eyebrow, blusher, eye cream, eye shadow, mascara, eyeliner, lipstick, lip balm, shampoo, rinse, Examples include hair dyes, dispersions, and cleaning materials. Examples of the form of a pharmaceutical or quasi-drug that can contain the hyaluronidase inhibitor of the present invention include ointments, creams, and liquids for external use.

  In addition to the hyaluronidase inhibitor according to the present invention, the external preparation for skin of the above-described form includes ingredients, oils, higher alcohols, and the like, which are blended in skin external preparations such as cosmetics and quasi-drugs, as long as the hyaluronidase inhibitory action is not impaired. Fatty acids, ultraviolet absorbers, powders, pigments, surfactants, polyhydric alcohols / sugars, polymers, physiologically active ingredients, solvents, antioxidants, fragrances, preservatives, and the like can be blended.

(2-2) Food etc. Since the composition of this invention can be ingested orally, it can also be utilized as food-drinks. In this case, the composition of the present invention can be eaten as it is or contained in an enteric capsule for administration. Or it can melt | dissolve in liquid (for example, water) so that it may become a suitable density | concentration, and it can add to foodstuff etc. by methods, such as mixing, immersion, application | coating, and spraying.

  In the food and drink of the present invention, the content ratio of the psyllium seed gum is not limited, and can be set as appropriate according to various uses. Moreover, when obtaining the food / beverage products of this invention, in the well-known manufacturing method according to various uses, a psyllium seed gum can be contained by arbitrary methods or timing. The form of the psyllium seed gum at the time of inclusion is not limited, and may be in the form of a slurry or powder, and may be appropriately selected according to various uses.

  The food / beverage products of the present invention are not particularly limited, and can include all foods that humans can eat. For example, edible oil (salad oil), confectionery (gum, candy, caramel, chocolate, cookies, snacks, jelly, gummy, tablet confectionery, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, Yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, tea, tea, carbonated beverages, sports drinks, etc.) and general foods, health foods (tablets, capsules, etc.), Nutritional supplements (such as energy drinks) are listed. The hyaluronidase inhibitor of the present invention may be appropriately blended with these foods and drinks.

  The addition amount of the composition of this invention to these food / beverage products can be selected according to the kind of food / beverage products used as the object of addition. However, if the amount added is too small, the expected hyaluronidase inhibitory effect cannot be sufficiently exhibited. On the other hand, if the amount added is too large, the flavor inherent to the food to be added may be impaired. Therefore, it is preferable to determine the addition amount in consideration of these circumstances. From this viewpoint, the amount of the composition added is about 0.01% to 20%, preferably about 0.01% to 5% of the mass of the food or drink to be added.

<Example 1>
Preparation of low-molecular psyllium seed gum 400 g of distilled water was added to 2 g of psyllium seed gum (manufactured by MRC Polysaccharide) and dissolved at room temperature (25 ° C.). 400 mL of the obtained aqueous solution was put into an ultrafilter, and the pressure difference between membranes was increased to 0.1 MPa using a commercially available nitrogen gas cylinder while stirring at 100 rpm using a magnetic stirrer. A stirred tank type ultrafiltration device (ADVANTEC-UHP76K) was used for the filter, and an ultracell PL (fractionated molecular weight: 100,000) was used for the membrane. After the pressurization, the ultrafiltration was terminated when the integrated permeation amount reached 300 mL. The obtained filtered solution was subjected to vacuum lyophilization to obtain 25 mg of low molecular weight psyllium seed gum.

Accurately weighed low molecular psyllium seed low molecular psyllium seed gum and psyllium seed gum 20mg unfractionated prepared as an average molecular weight measured above gum to prepare a sample solution as accurately 10mL with the mobile phase. Separately, 20 mg of a pullulan standard product (molecular weight 20,000) was accurately weighed and accurately adjusted to 20 mL with a mobile phase to obtain a standard solution. The sample solution and 300 μL of the standard solution were tested by gel permeation chromatography under the following operating conditions, and the average molecular weight of the sample was calculated.

Test conditions SIL-20AHT GPC system (SHIMAZU)
Detector: differential refractometer, light scattering photometer Column: OHpak SB-806M HQ (Shodex)
Column temperature: 40 ° C
Mobile phase: 50 mmol / L Na ₂ SO ₄ aqueous solution flow rate: 1.0 mL / min.

  The results are shown in FIG. The weight average molecular weight of the low molecular psyllium seed gum was 46966, and the weight average molecular weight of the unfractionated psyllium seed gum was 852950.

Hyaluronidase inhibitory activity test The hyaluronidase inhibitory activity of the low molecular psyllium seed gum was measured. 0.1 mL of different concentrations of psyllium seed gum (0.05 to 0.3 mg / mL) was added to 0.1 mL of hyaluronidase (3.5 mg / mL), respectively, and incubated at room temperature for 20 minutes. As the hyaluronidase, one separated from the testes of cattle (manufactured by Sigma) was used.

  Next, 0.2 mL of 0.1 mg / mL Compound 48/80 as a hyaluronidase activator and 0.5 mL of hyaluronic acid derived from human heso as a substrate (manufactured by Sigma) were added to this solution, and the mixture was heated at 37 ° C. for 40 minutes. Reacted for 1 minute. Thereafter, 0.2 mL of 0.4N NaOH was added to inactivate hyaluronidase and heated to 100 ° C., and then N- produced by a modified method of the Morgan-Elson method (J. Bio. Chem., 217, 959 (1955)). The amount of acetylhexosamine was determined from the absorbance OD 585 nm. In addition, 0.1 mM acetate buffer (pH 3.5) was used for the enzyme reaction.

  For each sample, 10-fold diluted concentrated sulfuric acid in which p-dimethylaminobenzaldehyde was dissolved was added to a solution in which hyaluronidase was inactivated, and incubated at 37 ° C. for 20 minutes. Then, the absorbance of this solution was measured at a wavelength of 585 nm. The inhibition rate was determined by the following equation.

A: Absorbance in the presence of inhibitor B: Absorbance in the absence of inhibitor C: Absorbance in the absence of enzyme

A similar inhibition test was performed using unfractionated psyllium seed gum without ultrafiltration as a control.

Next, 50% inhibition concentration (IC ₅₀ ) was determined from the inhibition rate of psyllium seed gum at various dilution concentrations, and the results are shown in FIG. The IC ₅₀ of the low molecular psyllium seed gum was 0.052 mg / mL, and the IC ₅₀ of the unfractionated psyllium seed gum was 0.081 mg / mL. The result is the average of 5 experiments. As can be seen from FIG. 2, strong hyaluronidase inhibitory activity was observed in the low-molecular psyllium seed gum. Therefore, it can be seen that the low-molecular psyllium seed gum having the effect of significantly suppressing this enzyme has a strong anti-inflammatory and anti-allergic action.

Claims (2)

A hyaluronidase inhibitor comprising , as an active ingredient , a low-molecular psyllium seed gum having a weight average molecular weight of 1000 to 100,000 measured by gel permeation chromatography . An antiallergic agent or anti-inflammatory agent comprising the hyaluronidase inhibitor according to claim 1.