JP2007230918A - Phospholipase a2 inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a phospholipase A2 inhibitor containing natural extracts. <P>SOLUTION: The present invention utilizes that extracts, obtained by extracting Arnica montana and Gentiana lutea with water, a hydrophilic organic solvent or a mixture thereof, have phospholipase A2 inhibitory activity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to phospholipase A ₂ inhibitors.

There are various causes and onset mechanisms of inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and various other skin diseases associated with rough skin. One of the causes is phospholipase A. _Two activations are known.

Phospholipase A ₂ is an important enzyme in the arachidonic acid cascade, which is the metabolic pathway of arachidonic acid. When phospholipase A ₂ is excessively activated, abnormal metabolism occurs in arachidonic acid, causing inflammation, allergy, asthma, ischemia, myocardium Causes infarction and the like. Therefore, attempts have been made to address the above-mentioned diseases by substances which inhibit the action of phospholipase A _2, as phospholipase A ₂ inhibitor, mepacrine, Parabromophenacyl bromide, dexamethasone and the like have been used, these All the substances were synthetic products, and side effects were a problem.

Therefore, by extracting a substance that inhibits phospholipase A ₂ from natural products, it has been attempted to prevent and treat allergic diseases diseases and inflammatory diseases. For example, a Keitanka extract (patent document 1), a kangsanfu extract (patent document 2), etc. are reported.
However, there is a strong demand from consumers for natural anti-inflammatory agents having high phospholipase A ₂ inhibitory activity, which are excellent in safety and productivity, and are inexpensive, and those that can still be fully satisfied are provided. There is no current situation.

JP 2002-97151 A JP 2002-53477 A

An object of the present invention is to find a natural product having a phospholipase A ₂ inhibitory activity, and to provide a phospholipase A ₂ inhibitor containing the same as an active ingredient.

As a result of intensive studies by the present inventors in order to solve the above problems, it has been found that extracts from Arnica and gentiana have a phospholipase A ₂ inhibitory action and are effective as phospholipase A ₂ inhibitors.

The present invention, phospholipase A ₂ inhibitor. Phospholipase A ₂ inhibitors of the present invention are useful for preventing and / or improving skin inflammation. Further, the present invention, a skin cosmetic and a food or drink having excellent safety having phospholipase A ₂ inhibitory action is provided.

  Arnica (scientific name: Arnica montana) used as a raw material for extraction is a perennial belonging to the family Asteraceae and grows on pastures of acidic soils in mountainous regions from Europe to Southern Russia and is easily available from these regions It is. The constituent parts of Arnica used as the extraction raw material are not particularly limited, and for example, constituent parts such as flower buds, leaf parts, branch parts, seeds, bark, and root parts can be used as the extraction raw material. Among these constituent parts, it is particularly preferable to use flowers as an extraction raw material.

  Moreover, gentian (scientific name: Gentiana lutea) used as an extraction raw material is a perennial plant that grows in European mountains and can be easily obtained from these regions. The constituent parts of gentian used as the extraction raw material are not limited to JP, but constituent parts such as florets, leaves, branches, seeds, bark, and roots can be used as the extraction raw material. Among these constituent parts, it is particularly preferable to use the root as an 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. Component showing the phospholipase A ₂ inhibitory action included in the extracted raw material, can be easily extracted by the extraction process of the polar solvent and the 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.

Although obtained extract can be used as it is as even phospholipase A ₂ inhibitors, towards those with concentrate or dried product is easily available. 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.

Extracts from Arnica and Gentian can be used as they are as phospholipase A ₂ inhibitors, but can also be provided after being formulated in accordance with conventional methods. 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.

Phospholipase A ₂ inhibitors of the present invention it is possible to prevent and / or improve the skin inflammation and excellent safety when applied to the skin, it is suitable for incorporation into skin cosmetics. The skin cosmetic, may be blended only phospholipase A ₂ inhibitor, it may be formulated by combining other active materials.

Skin cosmetics can be incorporated phospholipase A ₂ inhibitors of the present invention is not particularly limited, and specific examples thereof can be exemplified ointment, cream, milky lotion, lotion, pack, a bathing agent.

The amount of phospholipase A ₂ inhibitors of the present invention can be appropriately adjusted by the physiological activity and the like of the skin cosmetic of the type and extracts, preferred blending ratio is about in terms of standard extract 0 0.01 to 10% by mass.

The skin cosmetic containing the phospholipase A ₂ inhibitor of the present invention contains various main agents and auxiliaries usually used in the manufacture of skin cosmetics and other optional auxiliaries as long as they do not interfere with the phospholipase A ₂ inhibitory action. can be formulated, relates antiaging and improvement of skin, only phospholipase a ₂ inhibitors of the present invention is not limited to those comprising a main agent.

Phospholipase A ₂ inhibitors of the present invention because it is possible to prevent and / or ameliorate inflammation of the skin, has excellent safety, it is suitable for incorporation into food products. 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.

A food or drink can be incorporated phospholipase A ₂ inhibitors of the present invention is not particularly limited, and specific examples thereof include soft drinks, carbonated drinks, energy drinks, fruit drinks, beverages such as lactic acid beverage (concentration stock solution of these beverages And 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 .; fishery and livestock 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 Seasoning; soups, stews, salads, prepared foods, pickles, and the like.

Phospholipase A ₂ inhibitors of the present invention described above is intended to be suitably applied to human, as long as the respective actions and effects are exhibited, can also be applied to animals other than humans.

  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]
1000 ml of water, 50% ethanol or ethanol as an extraction solvent was added to 100 g of each of Arnica and gentian coarsely pulverized products, followed by extraction with a reflux extractor at 80 ° C. for 2 hours and filtration. 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 (%)
Arnica 8.4 6.0 4.2
Gentian 5.8 3.7 2.2

[Test Example 1] Phospholipase A ₂ inhibition test RBL-2H3 cells of rat leukemia cells were cultured in a 75 cm ² flask in MEM medium containing 15 v / v% FBS at 37 ° C. under 5% CO ₂ -95% air. Cells were collected by conventional methods. The obtained cells were prepared to 5 × 10 ⁵ cells / mL in MEM medium containing 15 v / v% FBS, and [ ³ H] arachidonic acid (50 μCi / 500 μL) was further added at a rate of 3 μL / 10 mL. . 1 mL of the obtained preparation solution was inoculated on a 24-well plate and cultured overnight at 37 ° C. under 5% CO ₂ 95% air. The medium in each well was discarded, and after washing with PBS (−), serum-free MEM medium was added and incubated at 37 ° C. for 30 minutes. Then, the solution in which the sample was dissolved was added to each well, and incubated for 10 minutes in the same manner. Further, 10 μL of 1 mmol / L A23187 was added and incubated at 37 ° C. for 5 minutes. After the reaction, 500 μL of the supernatant was taken under water cooling, 6 mL of scintillation cocktail was added, and the radioactivity was measured with a liquid scintillation counter. Similarly, measured for the blank test (A23187 no stimulation) and control (solvent of the sample solution), it was determined phospholipase A ₂ activity inhibition ratio by the following equation.
Inhibition rate (%) = [(BA) / (BC)] × 100
A: Radioactivity at the time of sample addition B: Radioactivity of the control C: The radioactivity sample concentration in the blank test was decreased stepwise to perform the above measurement, and the sample concentration IC ₅₀ (ppm) at which the inhibition rate was 50% was determined. Obtained by interpolation.

[Table 2] Phospholipase A ₂ inhibitory action
Test sample IC ₅₀ (ppm)
Arnica water extract 76.5
Arnica 50% ethanol extract 27.6
Arnica ethanol extract 68.6
Gentian water extract 198.3
Gentian 50% ethanol extract 185.1
Gentian ethanol extract 192.4
As shown in Table 2, it extracts from arnica and gentian is to exhibit a concentration dependent manner phospholipase A ₂ inhibitory action was confirmed.

Claims (1)

A phospholipase A2 inhibitor comprising at least one extract of Arnica and Gentian as active ingredients.