JP2007230915A - Hyaluronidase activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hyaluronidase activity inhibitor containing natural extracts. <P>SOLUTION: The present invention utilizes that extracts, obtained by extracting Thymus serpyllum and Humulus lupulus with water, a hydrophilic organic solvent or a mixture thereof, have hyaluronidase inhibitory activity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a hyaluronidase activity inhibitor.

  There are various causes and origins of inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other types of skin diseases. Causes of this are caused by hyaluronidase. It has been known.

  The hyaluronidase is an enzyme that hydrolyzes hyaluronic acid, but hyaluronic acid is a mucopolysaccharide found in the mesenchymal tissue and serves to prevent invasion and transmission of microorganisms and toxicants. When activated, the damage caused by the decrease in the protective effect of hyaluronic acid is likely to occur. Hyaluronidase is also related to type I allergic reaction and is considered to be an enzyme that controls degranulation from mast cells. Therefore, the enhancement of moisture retention or anti-inflammatory action can be expected by stabilizing hyaluronic acid and preventing the release of various chemical mediators from mast cells.

  Therefore, attempts have been made to extract substances having such a hyaluronidase inhibitory activity from natural products. For example, extracts of plants belonging to the genus Osbeckia (see Patent Document 1), Fuji tea extract (see Patent Document 2). Rosemary, Thyme and Melissa extract (see Patent Document 3) have been reported.

Japanese Patent Laid-Open No. 2003-55242 JP 2003-12532 A JP-A-8-333267

  An object of the present invention is to find a hyaluronidase activity inhibitory action from natural products, and to provide a hyaluronidase activity inhibitor containing the same as an active ingredient.

  As a result of intensive studies by the present inventors in order to solve the above-mentioned problems, it has been found that wild time, hop extract has a hyaluronidase activity inhibitory action and is effective as a hyaluronidase activity inhibitor.

  According to the present invention, a hyaluronidase activity inhibitor is provided. The hyaluronidase activity inhibitor of the present invention is useful for preventing and / or ameliorating skin inflammation.

  Moreover, the skin cosmetics and food / beverage products which have the hyaluronidase activity inhibitory effect and excellent in safety | security by this invention are provided.

  In the present invention, wild time (scientific name: Thymus serpylum) belongs to the family Lamiaceae, is native to Europe and Northwest Asia, has been introduced to other regions, has become naturalized, and can be easily obtained from these regions. is there. The constituent part of the wild time used as the extraction raw material is not particularly limited, but it is preferable to use the above-ground part as the extraction raw material.

  Hops (scientific name: Humulus lupulus) belong to the mulberry family, are native to Europe from western Asia and are readily available from these regions. The constituent part of the hop used as the extraction raw material is not particularly limited, but it is preferable to use female flower spikes as the extraction raw material.

  The said plant used as an extraction raw material can be obtained by drying and grinding | pulverizing as it is using a crusher, and using for solvent extraction. Drying may be performed in the sun or using a commonly used dryer. Each extraction raw material may be used as an extraction raw material after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. By performing a pretreatment such as degreasing, the extraction raw material can be efficiently extracted with a polar solvent. In the extraction process, it is preferable to use a polar solvent as the extraction solvent. A component having an inhibitory action on hyaluronidase activity contained in the extraction raw material can be easily extracted by an extraction process using a polar solvent as an extraction solvent.

  Specific examples of suitable extraction solvents include water, lower aliphatic alcohols, hydrous lower aliphatic alcohols, and the like, and these can be used alone or as a mixture of two or more thereof. Specific examples of suitable lower aliphatic alcohols include methanol, ethanol, propanol, 1,3-butylene glycol, glycerin, propylene glycol and the like.

  The water that can be used as the extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using the liquid mixture of water and a lower aliphatic alcohol, the mixing ratio of water and a lower aliphatic alcohol can be set to 9: 1 to 1: 9 (mass ratio).

  The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. In the extraction process, it is not necessary to adopt a special extraction method, and any apparatus can be used at room temperature or under reflux heating.

  For example, the extraction raw material is put into a treatment tank filled with the extraction solvent, and the soluble components are eluted with occasional stirring. At this time, the extraction conditions can be appropriately adjusted according to the extraction raw material, etc., but the amount of the extraction solvent is usually 5 to 15 times (mass ratio) of the extraction raw material, the extraction time is usually 1 to 3 hours, and the extraction temperature is Usually, it is normal temperature to 95 ° C.

  An eluate can be obtained by eluting soluble components by extraction and then filtering to remove the extraction residue. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed.

  The obtained extract can be used as a hyaluronidase activity inhibitor as it is, but a concentrated solution or a dried product thereof is easier to use. In addition, since the plant that is the raw material for extraction has a unique odor, it can be purified for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in its physiological activity. In this case, since it is not used in large quantities, there is no practical problem even if it is not purified.

  Specifically, purification can be performed by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like.

  The extract from wild time and hop can be used as it is as an inhibitor of hyaluronidase activity, but it can also be provided after being formulated according to a conventional method. In the case of formulating, a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and other optional auxiliaries can be added to facilitate storage and handling.

  The hyaluronidase activity inhibitor of the present invention can prevent and / or ameliorate skin inflammation and is excellent in safety when applied to the skin, and thus is suitable for blending into skin cosmetics. Only a hyaluronidase activity inhibitor may be mix | blended with skin cosmetics, and you may mix | blend in combination with another active material.

  Although the skin cosmetics which can mix | blend the hyaluronidase activity inhibitor of this invention are not specifically limited, As an example, an ointment, cream, milky lotion, a pack, a bath agent etc. can be illustrated.

  The blending amount of the hyaluronidase activity inhibitor of the present invention can be adjusted as appropriate depending on the type of skin cosmetic, the physiological activity of the extract, and the like. 01 to 10% by mass.

  The skin cosmetics containing the hyaluronidase activity inhibitor of the present invention should be blended with various main agents and auxiliaries usually used in the production of skin cosmetics and other optional auxiliaries as long as they do not interfere with the hyaluronidase activity inhibitory action. In terms of preventing and improving skin aging, the hyaluronidase activity inhibitor of the present invention is not the only main ingredient.

  Since the hyaluronidase activity inhibitor of the present invention can prevent and / or improve skin inflammation and is excellent in safety, it is suitable for blending into foods and drinks. In this case, it is appropriate that the amount of the extract per day for adults is about 1 to 1000 mg in consideration of the general intake of the food and drink to be added.

  Foods and drinks that can contain the hyaluronidase activity inhibitor of the present invention are not particularly limited, and specific examples thereof include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (concentrated stock solutions of these drinks and Including ice cream, ice sherbet, shaved ice, etc .; noodles such as buckwheat, udon, harusame, gyoza skin, cucumber skin, Chinese noodles, instant noodles; rice cake, chewing gum, candy, gum, chocolate , Tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, etc .; fish and shellfish processed foods such as kamaboko, ham, sausage; dairy products such as processed milk, fermented milk; salad oil, tempura oil, margarine , Mayonnaise, shortening, whipped cream, dressing and other fats and oils processed foods; sauce, sauce Seasonings such as soups, stews, salads, prepared dishes, and pickles.

  The hyaluronidase activity inhibitor of the present invention described above is suitably applied to humans, but can also be applied to animals other than humans as long as the respective effects are exhibited.

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

[Production Example 1]
100 g each of wild time and hop coarsely pulverized products was added as an extraction solvent, and 1000 ml of water, 50% ethanol or ethanol was added, extracted at 80 ° C. for 2 hours with a reflux extractor, and filtered. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and dried to obtain the extract. The yield of each extract is shown in Table 1.

[Table 1] Yield of extract (%)
Raw material water (%) 50% ethanol (%) Ethanol (%)
Wild time 8.6 5.5 3.2
Hop 40.3 34.8 28.6

[Test Example 1] Hyaluronidase inhibitory activity 0.1 mL of a hyaluronidase solution (400 units / mL, pH 3.5 acetate buffer) and 0.2 mL of a sample solution were mixed, incubated at 37 ° C for 20 minutes, and then an activator solution ( The enzyme was activated by adding 0.2 mL of 2.5 mM CaCl ₂ ) and incubating at 37 ° C. for 20 minutes. After adding 0.5 mL of potassium hyaluronate buffer and incubating at 37 ° C. for 40 minutes, 0.2 mL of 0.4N sodium hydroxide was added and ice-cooled to stop the reaction. Next, 0.2 mL of 0.8 M boric acid solution (pH 9.1) was added, heated in a boiling bath for 3 minutes, and immediately cooled on ice for 20 minutes. p-DABA reagent (10 g of p-dimethylaminobenzaldehyde dissolved in a mixture of 12.5 mL of 10N hydrochloric acid and 87.5 mL of acetic acid and diluted 10-fold with acetic acid) was added to 6.0 mL and incubated at 37 ° C. for 20 minutes. As a result, N-acetylglucosamine released by the enzyme reaction was colored and the absorbance at a wavelength of 585 nm was measured. The same operation and absorbance measurement were performed without adding the enzyme. Further, the same measurement was performed when distilled water was added without adding the sample solution, and the inhibition rate of hyaluronidase was determined by the following equation.
Inhibition rate (%) = {1− (A−B) / (C−D)} × 100
A: Absorbance when enzyme is added and sample solution is added B: Absorbance when enzyme is not added and sample solution is added C: Absorbance when enzyme is added and sample solution is not added D: Absorbance when enzyme is not added and sample solution is not added Sample concentration The inhibition rate was measured stepwise and the sample concentration (ppm) at which the inhibition rate was 50% was determined by interpolation.

[Table 2] Hyaluronidase activity inhibitory action
Test sample IC ₅₀ (ppm)
Wild Time Water Extract 542.2
Wild time 50% ethanol extract 258.4
Wild Time Ethanol Extract 443.2
Hops / water extract 578.1
Hops 50% ethanol extract 390.0
Hop ethanol extract 551.6

As shown in Table 2, it was confirmed that extracts from wild thyme and hops showed a hyaluronidase activity inhibitory effect in a concentration-dependent manner.

Claims (1)

A hyaluronidase activity inhibitor comprising at least one or more wild thyme and hop extracts as active ingredients.