JP2005239559A - Phospholipase a2 inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a PLA2 (phospholipase A2) inhibitor expecting effects such as suppression of edema formation and reduction of a pruritic feeling due to antiinflammation by inhibitory effects on the PLA2 and suitably usable in applications such as medicines, cosmetics or foods. <P>SOLUTION: The PLA2 inhibitor comprises an extract obtained from pine bark as an active ingredient, i.e. the PLA2 inhibitor comprises a resin ingredient extracted from the pine bark with a polar solvent as the active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a phospholipase A2 inhibitor comprising an extract obtained from pine bark as an active ingredient.

The onset mechanism of edema caused by inflammation is due to the migration and invasion of inflammatory cells to release inflammatory substances due to inflammation. Examples of the inflammatory substance include histamine, leukotriene, prostaglandin, and kinin-based substance. Histamine dilates blood vessels and increases vascular permeability. As a result, not only protein components but also various substances and cellular components ooze into tissues, and neutrophils and macrophages gather in inflammatory cells due to the concentration gradient of anaphyfilatoxin (LTB4, C5a, etc.). It is said that edema occurs.
Phospholipase A2 (hereinafter abbreviated as PLA2), which is the initiation enzyme of the arachidonic acid cascade, is an important enzyme. The reason for this is that arachidonic acid is bound to the 2-position of membrane phospholipid, and arachidonic acid cleaved after being hydrolyzed by PLA2 can be used for various enzymes such as prostaglandins, thromboxanes, leukotrienes and lipoxins. It is converted into other physiologically active substances, and inflammatory mediators such as leukotrienes and prostaglandins are also produced.
When the arachidonic acid cascade is activated, it is said that the prostaglandins that are produced specifically increase local blood flow, enhance edema formation and inflammatory cell invasion, and promote inflammatory responses.
Therefore, it is said that the production of these inflammatory mediators such as histamine, leukotriene, prostaglandin causes inflammation and causes edema or pruritus (itch).
(Standard Pharmacology No. 28, p. 286: Non-patent document 1 :)
Therefore, suppressing the action of the arachidonic acid cascade suppresses the occurrence of edema and the occurrence of pruritus, and an effective PLA2 inhibitor has been demanded from that point.
Standard Pharmacology 28, 286

An object of the present invention is to provide a PLA2 inhibitor composed of a pine bark extract, which can be expected to be effective for use in medicines, cosmetics, foods, etc. There is to do.

As a result of intensive studies in view of the above problems, the present inventors have obtained the knowledge that the resin component extracted from pine bark as a raw material has a remarkable effect as an inhibitor of PLA2, and complete the present invention. It came to. That is, the present invention is the following [1].
[1] A phospholipase A2 inhibitor comprising an extract obtained from pine bark as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the phospholipase A2 inhibitor which uses the resin component extracted from the pine bark using the polar solvent as an active ingredient is provided. Due to the effect of inhibiting phospholipase A2, anti-inflammatory effects such as suppression of edema generation and reduction of pruritus can be expected, and it can be suitably used for applications such as pharmaceuticals, cosmetics and foods.

The PLA2 inhibitor of the present invention comprises an extract obtained from pine bark as an active ingredient.
Here, the PLA2 inhibitor used in the present invention is a resin component extracted using pine bark as a raw material, and the pine used as a raw material may be a plant belonging to the genus Pinus, particularly in the type and country of origin. It is not limited. Specific examples include red pine, black pine, spruce pine, goyo pine and the like. Furthermore, for example, French coastal pine is preferable, and it is preferable to use wild species of French coastal pine, or horticultural pine obtained by crossing these. The raw material is preferably a pine bark portion.

The resin component extracted from the pine bark used in the present invention is extracted using a pine bark portion and a mixed solvent with a polar solvent such as water or ethanol. The extraction temperature is not particularly limited and can be appropriately selected, but may be any temperature from room temperature to the boiling point of the solvent. For example, about 50-70 degreeC is mentioned as conditions with more preferable extraction temperature conditions.
Furthermore, the method of obtaining the pine bark extract used for this invention is specifically manufactured as follows, for example. The raw pine bark is pulverized, and bark pieces are extracted with a mixed solution of water and ethanol at about 50 to 70 ° C. This is filtered to obtain an extract. This extract is salted out using an inorganic salt such as salt to remove tannins from the polymer fraction. Filtration is again performed to obtain an extract containing a large amount of proanthocyanidins having a molecular weight of 300 to 2500.
Proanthocyanidins have been proposed to be compounds that produce anthocyanidins by cleavage of their carbon-carbon bonds. And cyanidin is one of anthocyanidins and is widely present as a glycoside. When a compound is heated with an acid treatment, the carbon-carbon bond is cleaved to generate cyanidin, which is called procyanidin. Most of the naturally occurring proanthocyanidins are of the procyanidin type. Among these, OPC (oligomeric proanthocyanidins) is a condensed tannin having a relatively low molecular weight centered on a dimer to tetramer of catechin, and is flavan-3-ol or flavan-3, 4 A compound in which a diol is used as a structural unit and is bonded by condensation or polymerization.

As the proanthocyanidins having a molecular weight of 300 to 2500 thus obtained, for example, a column temperature of 30 ° C. and a flow rate of 1. with a 10 mM phosphate buffer (pH 2.6) and acetonitrile (93: 7) in a C18 reverse phase column. When fractionation is performed at 0 ml / min and analysis is performed at an absorbance of UV 280 nm, catechin, epicatechin, epicatechin gallate, procyanidol B1, procyanidol B2, procyanidol B3, procyanidol B4, toxifolin, etc. Is detected separately. 97% of the total extracts are these substances with a molecular weight of 300-2500. Examples of such products include commercial products manufactured by Toyo Shinyaku Co., Ltd., product name Flavangenol, product name Pycnogenol manufactured by Hofa Research, or product name Belpine manufactured by Berkem.
The extract may be appropriately concentrated to be used as a liquid extract, or the liquid extract may be powdered by a spray drying method. Furthermore, it is good also as a powder form by adding excipients, such as dextrin, to the said liquid extract, and spray-drying or freeze-drying. In particular, since stability over time can be obtained by solidifying and powdering, and from the viewpoint of handleability, the extract is more preferably solidified and powdered by freeze-drying.
These excipients include lactose, maltooligosaccharides, dextrins and the like. These excipients may be blended as a freeze-dried solidified product or powdered product, or may be blended into the concentrated liquid obtained as described above and powdered. The amount of the excipient is preferably spray-dried or freeze-dried by adding 10 to 70 parts by weight of the excipient to 100 parts by weight of the concentrated solution having a concentration of 8% by weight, for example.

For powdering, any method such as freeze-drying or spray-drying can be adopted. Of these, freeze-drying is more preferable. In this case, it is preferable to freeze-dry the above-mentioned concentrate or excipient added, and then pulverize using a blender for grinding. As for conditions such as freeze-drying, conditions usually used are appropriately selected. For example, the lyophilization temperature is preferably -3 to -40 ° C, particularly preferably about -10 to -30 ° C.

The form used as the PLA2 inhibitor of the present invention is not particularly limited, and for example, it can be used as a form of liquid, tablet, pill, granule powder, capsule or the like.
The PLA2 inhibitor of the present invention can be used by adding to a tableted product. In addition, foods to which the PLA2 inhibitor is added can be taken for the purpose of preventing or reducing inflammation such as suppression of edema and suppression of pruritus. The tableted product may be a pine extract crude purified product of proanthocyanidins, or a product obtained by blending an excipient as necessary.

Hereinafter, the present invention will be described in more detail with specific examples.
<Method for evaluating phospholipase A2 inhibitory effect>
In the present invention, pine lip extract was used to inhibit the phospholipase A2 activity using the following measurement method.
<Reagent>
1. Tris-HCl buffer (pH 8.0)
2-Amino-2-hydroxymethyl-1,3-propanediol [tris (hydroxymethyl) aminomethane] is dissolved in purified water to 0.01M and calcium chloride to 0.05M, and the pH is 8.0 with hydrochloric acid. Adjust to.
2. A solution of phosphatidylcholine having a purity of 70% or more dissolved in PC solution polyoxyethylene (10) octylphenyl ether (BioRad, trade name: Triton X-100) is filtered through a 0.45 μm filter.
3. Enzyme solution Phospholipase A2 (enzyme activity 2 IU / ml) solution is used.
4). This is a 0.1 M aqueous solution of a reaction stop solution EDTA · 2Na (ethylenediamine-N, N, N ′, N′-tetraacetic acid-2Na · 2H 2 O).
5). A predetermined amount of a sample for measuring inhibition of test solution activity is dissolved in Tris-NCl buffer. (The pine resin extract is dissolved in a Tris-HCl buffer solution by 0.4 to 4% (w / v), respectively.)

Next, a method for measuring the phospholipase A2 activity inhibitory effect will be described.
<Measurement operation method>
(1) Collect and mix 1 ml of the test solution and 0.4 ml of the enzyme solution in a 10 ml screw-cap test tube, and preheat for 5 minutes while shaking in a water bath adjusted to 37 ° C.
(2) Also, a test using 1 ml of Tris-HCl buffer instead of the test solution as a blank is performed in parallel. After 5 minutes, 0.6 ml of the PC solution is added and mixed, and the mixture is reacted for 15 minutes while shaking in a 37 ° C. water bath.
(3) After 15 minutes, 1.5 ml of the reaction stop solution is added and mixed to stop the reaction. Here, exactly 5 ml of chloroform was added to extract lipids. The collected chloroform is subjected to thin layer chromatography (TLC).
(4) At this time, TLC spotted 12 μl of each sample equally with chloroform.
(5) The TLC plate is normal silica gel, and the developing solvent is petroleum ether / diethyl ether / acetic acid = 80/20/1 (volume ratio). After the completion of the development, 50% (v / v) sulfuric acid is sprayed, and color development is performed by heating in a 105 ° C constant temperature bath. The intensity of the spot of the free fatty acid in the blank is defined as 100, and the percentage of the spot intensity of the free fatty acid in the system to which the sample solution is added to the blank is defined as the reaction rate (%). The value obtained by subtracting this from 100 is taken as the reaction inhibition rate (%).
Reaction inhibition rate (%) = [{(strength of blank fatty acid) − (strength of fatty acid in test solution)} / (strength of blank fatty acid)] × 100

Example 1
Weigh out 0.04 g of pine bark extract (trade name Verpine: 10% by weight of flavan-3-ol monomer and 74% by weight of oligomers of 2 to 4 and molecular weight of 300 to 2500) manufactured by Berkem. , 0.01M Tris-HCl buffer solution (pH 8.0) 10ml was dissolved to make a 0.4% solution. 1 ml of this solution and 0.4 ml of the above PLA2 enzyme (Novo Co., Ltd., trade name: Residase 2 IU / ml) solution were mixed in a screw test tube and preheated at 37 ° C. for 5 minutes. did. Subsequently, 0.6 ml of a solution obtained by dissolving high-purity phosphatidylcholine in Triton X-100 solution was added and mixed, and reacted at 37 ° C. for 15 minutes. Thereafter, 1.5 ml of a 0.1 M EDTA solution was added as a reaction stop solution to stop the reaction. This is referred to as a test solution. Lipids were extracted from the test solution with chloroform and subjected to thin layer chromatography using a developing solvent [petroleum ether / diethyl ether / acetic acid = 80/20/1 (volume ratio)]. After the development, analysis was performed by heating and coloring with 50% sulfuric acid.

Examples 2-4
As shown in Table 1, the same pine bark extract used in Example 1 was used, and its concentration was adjusted to 0.8% by weight, 2% by weight, and 4% by weight. Went. The results are shown in Table 1.

  As can be seen from Table 1, in Examples 1 to 4 of the present invention, the reaction inhibition rate was 18.6% in the order of pine bark concentration of 0.4 wt%, 0.8 wt%, 2 wt% and 4 wt%. 38.0%, 51.7% and 57.4%, indicating that the reaction inhibition rate depends on the concentration of the pine bark extract. Thus, the pine bark extract has an inhibitory effect on phospholipase A2 activity, and is expected to have effects such as suppression of edema generation and reduction of pruritus as described above.

Reference Example 1 (Preparation of tablets)
2 g (2% by weight) of pine bark extract used in Example 1, 96 g (96% by weight) of dried corn starch [Nippon Food Chemical Co., Ltd.], 1.8 g of talc [Wako Pure Chemical Industries, Ltd.] (1. 8 wt%) and calcium stearate [Nippon Yushi Co., Ltd.] 0.2 g (0.2 wt%) were mixed and mixed with a rocking mixer for 10 minutes, and then 1 tablet, 0.52 g of the tablet using a tableting machine. Tablets were made.

From this, the pine bark extract which has the phospholipase A2 activity inhibitory effect can be made into the shape which is easy to ingest as a tablet.

Claims (1)

  A phospholipase A2 inhibitor comprising an extract obtained from pine bark as an active ingredient.