JPH11209299A - Cyclic amp phosphodiesterase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cyclic AMP phosphodiesterase inhibitor capable of inhibiting the coagulation of platelets from a naturally originated material and provide it as a food material useful for maintaining and promoting health. SOLUTION: This cyclic AMP phosphodiesterase inhibitor is obtained by utilizing an extract obtained by the extracting treatment of a plant belonging to the genus Bergenia such as Bergenia crassifolia and Bergenia purpirascens with water or a hydrophilic organic solvent or their mixtures, which has the cyclic AMP phosphodiesterase inhibiting action and also inhibits the coagulation of platelets.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cyclic AMP having a function of inhibiting the action of cyclic AMP phosphodiesterase, which degrades cyclic AMP, which is an important signal transmitter in vivo, and thereby inhibiting the aggregation of platelets. It relates to an AMP phosphodiesterase inhibitor.

[0002]

2. Description of the Related Art There are various causes for the formation of thrombus in capillaries, one of which is due to aggregation of platelets. In other words, when blood vessels rupture or endothelial cells detach due to inflammation due to exogenous compression such as bruising, or other various stimuli, platelets are activated by a signaling substance from endothelial cells, which aggregates This produces a thrombus (platelet thrombus).

[0003] Aggregation of platelets causing thrombus is related to the concentration of cyclic AMP in platelets, and cyclic AMP is generated by cyclic AMP phosphodiesterase.
When the concentration of cyclic AMP decreases due to MP degradation or the like, platelets tend to aggregate. Therefore, if the action of cyclic AMP phosphodiesterase is suppressed to prevent a decrease in cyclic AMP concentration, the generation of platelet thrombus should be able to be prevented. However, when examining the effect using various cyclic AMP phosphodiesterase inhibitors, it was found that cyclic A
Even though the MP concentration can be maintained properly, platelet aggregation cannot often be prevented. For unknown reasons, certain cyclic AMP phosphodiesterase inhibitors not only inhibit the action of cyclic AMP phosphodiesterase to prevent the degradation of cyclic AMP, but also act on platelets by another mechanism. It may be promoting aggregation.

[0004] Therefore, it is not always possible to suppress platelet aggregation and prevent platelet thrombus by a substance having a cyclic AMP phosphodiesterase inhibitory action.

[0005] For example, as extracts having a cyclic AMP phosphodiesterase inhibitory activity, there are known extracts of soybean arbor, chile, and the like. Among them, only the soybean arbor extract can inhibit platelet aggregation.

[0006]

Cyclic AMP is involved not only in platelet aggregation but also in fat metabolism and several other important biological events, but platelet aggregation only causes thrombus formation. Furthermore, the suppression is the most important function. Therefore, it is desirable that the cyclic AMP phosphodiesterase inhibitor not only inhibits the action of cyclic AMP phosphodiesterase but also suppresses the aggregation of platelets even if it is not its own direct action.

[0007] Therefore, the present invention provides a cyclic A which can inhibit platelet aggregation from naturally occurring substances derived from natural products.
An object of the present invention is to find an MP phosphodiesterase inhibitor and provide it as a food material effective for maintaining and improving health.

[0008] Another object of the present invention is to provide a cyclic AMP phosphodiesterase inhibitor which is suitable for use in an external preparation for skin in anticipation of a slimming effect by promoting metabolism of fat.

[0009]

Means for Solving the Problems The cyclic AMP phosphodiesterase inhibitor provided by the present invention extracts water, a hydrophilic organic solvent, or a mixture thereof from plants of the genus Himalaya, such as Bergenia classifolia and Bergenia perprasens. The extract obtained as a solvent is used as an active ingredient.

[0010] The cyclic AMP phosphodiesterase inhibitor inhibits the action of cyclic AMP phosphodiesterase and can further inhibit platelet aggregation.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Bergenia crassifolia (Japanese name: Naganoyukinosita) and Bergenia purpurasc
(ens) is a plant belonging to the genus Saxifragaceae in the genus Himalayan Saxifragaceae. It is said to be effective against frail constitution, hemoptysis, hemoptysis, inflammatory swelling, etc., and uses whole plants. In addition, there are regions that use it as tea (Heibonsha, World Dictionary of Useful Plants). However,
It was not known that this plant contained a substance that inhibits the action of cyclic AMP phosphodiesterase.

The cyclic AMP phosphodiesterase inhibitor is contained in a large amount in the root portion of the plant of the genus Himalaya. Extraction of the active ingredient is desirably performed on a dried product, and fresh one has poor extraction efficiency. As the extraction solvent, water, a hydrophilic organic solvent or a mixture thereof can be used. As the hydrophilic organic solvent, lower aliphatic alcohols such as methanol and ethanol are preferable. When a hydrophobic organic solvent is used, not only the extract yield is extremely low, but also the extract has a weak cyclic AMP phosphodiesterase inhibitory action.

The extraction method is not limited, and the extraction can be carried out at room temperature or under reflux with any apparatus. Briefly, an extraction raw material is charged into a treatment tank filled with an extraction solvent, and a soluble component is eluted with occasional stirring. When the obtained extract is concentrated and dried, cyclic AMP is obtained.
An extract having the action of inhibiting phosphodiesterase and inhibiting platelet aggregation is obtained.

This extract is used as it is for cyclic AMP.
It can be used as a phosphodiesterase inhibitor, but if necessary, it may be subjected to a purification treatment effective for improving its activity or removing coloring, unpleasant odor, off-taste, etc., and may be used in powders, granules, tablets, capsules, syrups, and intestines. It can be formulated into an arbitrary form such as a solubilizer, a troche, a chewable and the like, and used.

Extracts having a cyclic AMP phosphodiesterase inhibitory activity can also be obtained from other plants of the genus Himalayan Saxifrage, such as Bergenia syriata, Bergenia cordifolia, Bergenia ligrata, etc., but their importance as raw material plants is low.

The cyclic AMP phosphodiesterase inhibitor of the present invention comprising the above-mentioned extract has been confirmed to be safe, since the raw material of the genus Himalayan Saxifraga has been used for medicinal purposes since ancient times as described above. From
It can be used in foods and drinks and cosmetics without the risk of side effects. In other words, it can be added to any food or drink with the expectation of a fat metabolism-promoting effect, an antithrombotic effect, etc., so that its useful effects can be used at daily eating and drinking occasions. Can be added to creams, lotions, milky lotions, etc. with the expectation of a subcutaneous fat reduction effect.

The extract of the plant of the genus Himalayan Saxifrage has a light brown color, and when added to foods and drinks, does not significantly change the original flavor or properties of the foods or drinks, and changes its cyclic AMP phosphodiesterase inhibitory activity. It is not necessarily a general food and drink, such as seasonings,
For producing Japanese and Western confectionery, frozen desserts, soft drinks, tea, spreads, pastes, pickles, processed meat, processed marine products, dairy products, processed eggs, processed vegetables and fruits, processed cereals, etc. Auxiliary raw materials and additives used such as glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl -α-tocopherol,
Sodium erythorbic acid, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B, nicotinamide, pantothenic acid It can be added together with calcium, amino acids, calcium salts, pigments, flavors, preservatives and the like.

The preferred addition level varies depending on the quality and the substance to be added, but it is usually 0.01 to 10% by weight when a standard extract of the genus Himalaya is used.

When incorporated into cosmetics, various materials commonly used in the production of cosmetics such as fats and oils such as vegetable oils, waxes such as laurin and beeswax, hydrocarbons, fatty acids, higher alcohols, esters, It can be used in any combination with surfactants, pigments, fragrances, vitamins, plant / animal extract components, ultraviolet absorbers, antioxidants, preservatives / bactericides and the like.

[0020]

The present invention will be described below with reference to examples. In each example, “parts” means parts by weight.

Production Example 1 500 g of an extraction solvent was added to 100 g of dried root of Bergenia classifolia, and the mixture was heated under reflux for 1 hour to extract a cyclic AMP phosphodiesterase inhibitor twice. The obtained extracts are combined, concentrated under reduced pressure, and further dried to obtain an extract. Table 1 summarizes the results of the above-described extraction experiments performed with the six types of extraction solvents and the results of the same extraction performed on Bergenia perprusens.

[0022]

TABLE 1 Extraction yield (wt%) Extraction solvent B · crush cordifolia extract B · Papurasensu extract water 16.8 17.3 50% ethanol 21.5 20.8 Ethanol 28.0 27.1 Ethyl acetate 8.6 9.5 Acetone 5.3 6.5 n-Hexane 3.2 3.8

Test Example 1 A part of the extract of the plant belonging to the genus Himalayan safflower obtained in Production Example 1 was subjected to cyclic AM by the following test method.
The P phosphodiesterase inhibitory action was confirmed.

[0024] Test Method: Tris-HCl buffer containing MgCl ₂ and (pH7.5) 0.2ml, BSA solution 0.1ml and cyclic AMP phosphodiesterase solution Tris-HCl containing no MgCl ₂ in a mixture of 0.1ml Add 0.05 ml of a solution of the sample in buffer and preincubate at 37 ° C for 5 minutes. Then add this to cyclic AM
Add 0.05 ml of P solution and react at 37 ° C. for 30 minutes. After boiling for 3 minutes in boiling water, stop the reaction.
After centrifugation at 5 ° C., the reaction product.5′-AMP in the supernatant is quantified by high performance liquid chromatography. The same enzyme reaction and reaction product analysis are performed without adding the sample, and the cyclic AMP phosphodiesterase inhibition rate of the sample is determined from the ratio of the amount of the reaction product when the sample is added to the amount of the reaction product when the sample is not added.

The above measurement was carried out by changing the sample concentration of the sample solution to be added to the reaction solution stepwise, and the enzyme activity was reduced to 50%.
The inhibitory sample concentration (ppm) was determined by interpolation. Table 2 shows the results.

[0026]

50% inhibitory concentration (ppm) B. classifolia extract B. perplacens extract water extract 132.5 127.0 50% ethanol extract 54.2 52.2 ethanol extract 47.6 48. 6

Test Example 2 For the sample in which the cyclic AMP phosphodiesterase inhibitory effect was confirmed in Test Example 1, the platelet aggregation inhibitory effect was further tested by the following method.

Test method: One tenth volume of 77 mM EDTA was added to the blood of Japanese white rabbits, and the precipitate was removed by centrifugation at 1000 rpm for 10 minutes. The supernatant is centrifuged at 2100 rpm for 10 minutes, and the precipitated platelets are collected. The obtained platelets are suspended in a platelet washing solution and centrifuged at 2100 rpm for 10 minutes. The precipitated platelets were collected and the platelet count was 300,000 / μ.
Suspend the platelet suspension to l. 1 μl of a CaCl ₂ solution is added to 223 μl of the obtained washed platelet suspension, and the mixture is kept at 37 ° C. for 1 minute. Then, 1 μl of a sample solution is added and the mixture is kept at 37 ° C. for 3 minutes. Next, while maintaining the same temperature, 25 μl of the collagen solution is added, and the visible light transmittance is continuously measured for 10 minutes. The maximum visible light transmittance A measured during that time is used as an index of the platelet aggregation inhibitory effect. Separately, in the case where only the solvent for the sample solution is added instead of the sample solution, the same measurement as described above is performed, and the visible light transmittance B is measured. From the measured visible light transmittance, the platelet aggregation suppression rate is determined by the following equation. Platelet aggregation inhibition rate (%) = [(BA) / B] × 100

The above measurement was carried out while changing the concentration of the sample solution stepwise, and the results of the interpolation method were used to determine the concentration of the sample solution at which the platelet aggregation inhibition rate was 50%. Table 3 shows the results.

[0030]

[Table 3] Platelet aggregation 50% inhibitory concentration (ppm) B. classifolia extract B. purple extract extract water extract 80.5 79.5 50% ethanol extract 77.3 75.3 ethanol extract 94.2 96.7

Use Example 1 A lotion having the following composition and containing the ethanol extract of B. perprusens according to Production Example 1 was produced by a conventional method. B. purple extract ethanol extract 0.1 part glycerin 4 parts 1,3-butylene glycol 4 parts ethanol 7 parts polyoxyethylene oleyl alcohol 0.5 parts methyl paraben 0.05 parts citric acid 0.01 parts sodium citrate 0 .1 part Fragrance 0.05 part (total amount is 100 parts with purified water)

Use Example 2 A cream having the following composition and containing a 50% ethanol extract of B. classifolia according to Production Example 1 was produced by a conventional method. B. classifolia 50% ethanol extract 0.1 part Cetostearyl alcohol 3.5 parts Squalane 40 parts Beeswax 3 parts Reduced lanolyl 5 parts Ethyl paraben 0.3 parts Polyoxyethylene 2 parts Stearic acid monoglyceride 2 parts Fragrances 0.03 Part 1,3-butylene glycol 5 parts glycerin 5 parts (total amount is 100 parts with purified water)

Use Example 3 A chewing gum containing the ethanol extract of B. classifolia according to Production Example 1 and having the following composition was produced by a conventional method. B ・ classifolia ethanol extract 1 part chewing gum base 70 parts sucrose 54 parts starch syrup 20 parts softener 4 parts fragrance 1 part

Use Example 4 A chocolate having the following composition and containing 50% ethanol extract of B. perpulase according to Production Example 1 was produced by a conventional method. B. Purprasense 50% ethanol extract 0.1 part Sucrose 14 parts Cocoa butter 20 parts Whole milk powder 25 parts

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI A61K 7/00 A61K 7/00 W 7/48 7/48 // C12N 9/99 C12N 9/99 (72) Inventor Zhou Gloss Yo 14703-10 Mukoto-cho, Onomichi-shi, Hiroshima Prefecture Inside Maruzen Pharmaceutical Co., Ltd. (72) Inventor Shigetake Matsumoto 3-14-22 Kubo, Onomichi-shi, Hiroshima Prefecture

Claims (3)

[Claims] 1. A cyclic AMP phosphodiesterase inhibitor, which is effective for inhibiting platelet aggregation, comprising an extract obtained from a plant of the genus Himalayan Saxifrage using water, a hydrophilic organic solvent or a mixture thereof as an active ingredient. 2. The cyclic AMP phosphodiesterase inhibitor according to claim 1, wherein the plant of the genus Himalayan Saxifraga is Bergenia classifolia or Bergenia perpense. 3. The cyclic AMP phosphodiesterase inhibitor according to claim 1, wherein the extraction solvent is water, lower aliphatic alcohol or a mixture thereof.