JP2012025713A - Carpaine activity inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a carpaine activity inhibitor with excellent safety which can be utilized for the prevention and treatment of various diseases brought about by the surplus activation of carpaine.SOLUTION: It has been found out that one or more kinds of plants selected from the group consisting of the plants belonging to the Ericaceae Vaccinum and the plants belonging to the Rosaceae Eriobotrya or the solvent extract of these plants, particularly, Vaccinium myrtillus or the solvent extract thereof and Eriobotryae folium or its solvent extract have an remarkably excellent carpaine activity inhibiting action.

Description

  The present invention relates to a calpain activity inhibitor.

  Calpain is an intracellular proteolytic enzyme belonging to the cysteine protease group, and is found in many cells and tissues. Calpain is activated by increasing intracellular calcium concentration, calpain I or μ-calpain activated by μ-molar calcium ions, and calpain II or m activated by m-molar calcium ions. -Calpain is known and further calpain isozymes are envisaged.

  Calpain plays an important role in various physiological processes. Excessive activation of calpain is known to provide a molecular relationship between ischemia or injury induced by increased neuronal calcium and pathological neurodegeneration, such as cardiac ischemia, The onset of renal or central nervous system ischemia, inflammation, muscular dystrophy, cataract, diabetes, HIV disease, damage to the central nervous system, Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, etc. It has been pointed out that it is strongly involved.

  Further, since it has been reported that the release of interleukin-1α is blocked by a calpain inhibitor (Non-patent Document 1), calpain may be involved in the aging phenomenon of the skin. Therefore, calpain inhibitors are expected to be effective in delaying skin aging such as skin dryness, pigmentation, and wrinkle generation by suppressing the production of active oxygen such as superoxide anion by interleukin-1α. it can.

  Calpain inhibitors are broadly classified as irreversible and reversible inhibitors and peptide and non-peptide inhibitors. Specifically, specific peptides (Patent Document 1), α-substituted hydrazides (Patent Document 2), indole carboxamides (Patent Document 3), carboxamide compounds (Patent Document 4) and the like have been reported. However, since these calpain inhibitors may cause side effects, there are concerns in terms of safety, and the price is not satisfactory. In addition, no studies have been reported so far on inhibitors of calpain enzymes, which contain plant-derived components with few problems in terms of safety as active ingredients.

Japanese National Patent Publication No. 9-500087 Japanese National Patent Publication No. 11-500124 JP-T-2001-515508 Japanese Translation of PCT International Publication No. 2010-514738

N. Watanabe et al., Cytokine vol. 6 (6), 597-601 (1994)

  The present invention is to provide a calpain activity inhibitor excellent in safety.

  As a result of earnest research on a calpain activity inhibitor comprising a plant-derived component with less problems in terms of safety as an active ingredient, the present inventors have found that the bilberry extract and the koji leaf extract have a remarkably superior calpain activity inhibitory action. And the present invention has been completed.

That is, the present invention
[1] To provide a calpain activity inhibitor comprising as an active ingredient one or more kinds of plants selected from the group consisting of plants belonging to the genus Azalea family, genus Rosaceae, or a plant belonging to the genus Rosaceae To do.

  The plant belonging to the genus Azalea is bilberry, and the plant belonging to the genus Rosaceae is preferably a bamboo leaf. In the case of a solvent extract, it is extracted using ethanol, aqueous methanol or water. It is preferable that the extract obtained as described above is an active ingredient.

  [2] The calpain activity inhibitor may be an external preparation.

  Conventionally, ingredients that have been effective as calpain inhibitors may cause side effects, and there have been concerns in terms of safety. However, according to the present invention, various diseases caused by excessive activation of calpain can be obtained. It is possible to provide a calpain activity inhibitor with few problems in terms of safety that can be used for prevention and treatment.

It is a figure which shows the result of the activity measuring method (primary screening) using the electrophoresis method.

  Hereinafter, the configuration of the present invention will be described in more detail.

  The calpain activity inhibitor according to the present invention comprises a plant belonging to the genus Vaccinaceae genus or Viraceae, or a solvent extract of the plant as an active ingredient.

  The Rhododendron genus genus according to the present invention is an evergreen or deciduous shrub or tree that is widely distributed from the temperate zone to the cold zone of the northern hemisphere and about 300 species are known. Examples of the Azalea family include bilberry (Vaccinium myrtillus), blueberry (Vaccinium australe Small), cowberry (Vaccinium vitis-ideaea L.), blackberry (Vaccinium.uliginosum L.), cranberry (Vaccinium.macrocarpon Ait.), Etc. These can be used alone or in combination of two or more. The kind of the genus Vaccinium belonging to the present invention is not particularly limited, but bilberry (Vaccinium myrtillus) is preferable. The leaves, buds, bark, sap, fruits, etc. of the genus Vaccinaceae are medicinal and the use site is not particularly limited, but fruits containing anthocyanins are preferred.

  The plant belonging to the genus Rosaceae according to the present invention is an evergreen shrub or tree that is known for about 10 species distributed in the temperate region of East Asia. Examples of the Rosaceae Biwa genus plant include loquat (Eriobotrya japonica (Thunb.) Lindl.), Which is preferably used. There are no particular limitations on the site of use, but bamboo leaves known as bamboo leaves and components obtained from the leaves are preferred. Bamboo leaves contain oleanolic acid, ursolic acid, saponin, amygdalin, vitamin B1, tannin, and the like, and are sometimes used as crude drugs in the private sector.

  When the plant is used as an extract, the extraction solvent is not particularly limited, but the plant is a solvent, for example, a polar solvent such as water, lower alcohol, hydrous lower alcohol, propylene glycol, 1,3-butylene glycol, butanol, or chloroform. The extract obtained by extraction with an organic solvent such as ethyl acetate, various ethers or a mixture thereof is used as it is or after concentration, or the extract is adsorbed, for example, an ion exchange resin is used to remove impurities. It is also possible to use an extract that is adsorbed on a porous polymer column and then eluted with methanol or ethanol and concentrated. Also, a partitioning method, for example, an extract extracted with butanol can be used. However, in order to obtain a fraction having a higher calpain activity, extraction with ethanol, hydrous ethanol or water is preferred, and 50% hydrous ethanol is more preferred. As a city version product, for example, as an azalea family genus plant component, Phytelene of Bilberry EG 348 Liquid (bilberry berry / leaf extract) of Green Tech, Ichimaru Falcos Eco Farm Bilberry Leaf E, Cure Berry (both bilberry) Leaf extract), Blueberry extract B (bilberry extract) from Ikeda Sakuka Kogyo Co., Ltd., Kikkoman cranberry powder (cranberry juice) from Kikkoman, etc. Extract), Falco Rex Biwa Leaf B (bamboo leaf extract) from Ichimaru Falcos, Biwa Leaf Extract B (bamboo leaf extract) from Ikeda Sakuka Kogyo Co., Ltd., and so on. What was processed by the said method can also be utilized.

  The plant or plant solvent extract selected from the group consisting of the plant belonging to the genus Vaccinaceae and the plant belonging to the genus Rosaceae thus obtained has an excellent calpain activity inhibitory action, and has an excessive activity of calpain. It is useful for the prevention and treatment of various diseases caused by the conversion.

  In addition, extracts such as jujube leaves, which contain oleanolic acid and ursolic acid, such as jujube, forsythia, sanshuyu, perilla, ume, apricot and peach, may also have a calpain activity inhibitory effect. .

  The calpain activity inhibitor of the present invention is highly safe and can be administered by any method of internal use and external use, but is preferably used as an external preparation. The calpain activity inhibitor which is a form of the external preparation of the present invention can be blended with commonly used external base materials, medicinal ingredients and the like. External base materials include hydrocarbons such as liquid paraffin, vegetable oils and fats, waxes, synthetic ester oils, silicone oil phase components, fluorine oil phase components, higher alcohols, fatty acids, thickeners, UV absorption Agent, powder, pigment, color material, anionic surfactant, cationic surfactant, nonionic surfactant, amphoteric surfactant, polyhydric alcohol, sugar, polymer compound, promoting transdermal absorption An agent, a solvent, an antioxidant, a fragrance, a preservative, and the like can be arbitrarily blended. As the medicinal component, an analgesic / anti-inflammatory agent, bactericidal / disinfectant, vitamins, emollient and the like can be used as appropriate. The dosage form of the external preparation of the present invention is arbitrary, and any agent based on an oily base, an oil-in-water type, a water-in-oil emulsified base, or a water-based agent Any type can be used. Moreover, as a use, it can use suitably for cosmetics, a skin external preparation, a pharmaceutical ointment, etc. The form may take any form such as lotion, lotion, emulsion, cream, pack, ointment, dispersion, solid, mousse and the like. The dosage of this product is not particularly limited as long as it is within the normal range, but it is usually used in the range of 0.001 to 2000 mg as the active ingredient of calpain activity inhibitor per product per day. The compounding amount in the external preparation varies depending on the use, dosage form, compounding purpose, etc., and is not particularly limited, but is 0.001 to 20.0% by mass in the external preparation as the active ingredient of the calpain activity inhibitor, preferably Is used in the range of 0.01 to 10.0% by mass.

  In addition, as an external preparation, one or two or more kinds of plants selected from the group consisting of plants belonging to the genus Rhododendron belonging to the present invention, plants belonging to the genus Rosaceae, or a solvent extract of these plants as an active ingredient It has been found that the calpain activity inhibitor described above significantly suppresses the release of interleukin-1α (IL-1α) after ultraviolet irradiation. This suggests that the calpain activity inhibitor of the present invention inhibits the processing of calpain from Pro-IL-1α to IL-1α, which is a disease caused by an increase in IL-1α level. Expected to be useful for treatment.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

  Screening of plant extracts having calpain activity inhibitory action

1. Outline of Test Plant extracts having calpain activity inhibitory action were screened.

  2. experimental method

2-1. Activity measurement method using electrophoresis (primary screening)
To a 1.5 mL Eppendorf tube, a final concentration of 500 ng calpain I, each plant extract shown in Table 1, and a calcium-containing buffer were added. After incubation on ice for 10 minutes, a final concentration of 4 μg of β-casein was added, and the reaction was started at 37 ° C. After the reaction for 30 minutes, the reaction was immediately stopped by quenching on ice. SDS sample buffer was added to the reaction solution after stopping, and boiled at 100 ° C. for 5 minutes. The total amount of the reaction solution was developed with 12% SDS-PAGE, and the degradation degree of β-casein was detected by Coomassie Brilliant Blue staining by molecular weight comparison. In addition, the solution type plant extract was evaluated using the solid content obtained by freeze-drying among each plant extract.

2-2. Comparison of activity inhibition using a fluorescent substrate (D-casein-FITC) (secondary screening)
A 96-well plate was added with calpain I having a final concentration of 750 ng, among the plant extracts shown in Table 1, those that showed calpain inhibitory activity in the primary screening, and a calcium-containing buffer. After incubation on ice for 10 minutes, a final concentration of 2.5 μg of D-casein-FITC was added, and the reaction was started at 37 ° C. After reacting for 30 minutes, FITC fluorescence intensity change was measured with a fluorescence plate reader. In addition, the solution type plant extract was evaluated using the solid content obtained by freeze-drying among each plant extract.

3. Results An example of the results of screening for calpain inhibitory activity by electrophoresis is shown in FIG. By adding calpain I, a decrease in β-casein and an increase in the degradation product of β-casein are observed. Degradation suppression was observed.

Next, in order to compare the calpain activity inhibitory action, the 50% inhibitory concentration (IC50) of each plant extract in which the degradation suppressing action was recognized in the primary screening was evaluated. As a result, particularly high calpain inhibitory action was observed in the bilberry extract and the loquat extract (Table 2).

  Hereinafter, various preparations using the calpain activity inhibitor of the present invention were prepared, and examples are shown below. A compounding quantity is the mass%. In Examples 2 to 5, a calpain activity inhibitory action was observed.

(tablet)
Bilberry water / ethanol extract 5.0g
Corn starch 4.0g
Crystalline cellulose 30.0g
Lactose 10.0g
Talc 1.0g
Carboxymethylcellulose 5.0g
Light anhydrous silicic acid 0.5g
Magnesium stearate 0.5g
(Manufacturing method)
The above components were uniformly mixed, and tablets having a diameter of 8 mm and a weight of 180 mg were prepared according to a conventional method.

(Granule)
Biwayo water extract 10.0g
Lactose 70.0g
16.5g starch
Hydroxypropylcellulose 3.5g
(Manufacturing method)
Mix evenly and knead. After granulation by an extrusion granulator, it was dried and sieved to give granules.

(Ointment formulation)
Bilberry water / butylene glycol extract 10.0g
Polyoxyethylene cetyl ether 2.0g
Glyceryl monostearate 10.0g
Liquid paraffin 10.0g
White petrolatum 5.0g
Cetanol 6.0g
Propylene glycol 10.0g
47.0g of purified water
Preservative appropriate amount (production method)
According to a conventional method, they were mixed to obtain an ointment preparation.

(External preparation (emulsion))
Biwayo water / ethanol extract 15.0 g
White petrolatum 41.0g
Microcrystalline wax 3.0g
Lanolin 10.0g
4.75 g sorbitan monooleate
Monooleic acid POE sorbitan (20E.O.) 0.25 g
47.0g of purified water
Preservative appropriate amount (production method)
An emulsion composition was prepared according to a conventional method for producing an emulsion composition.

Claims (4)

A calpain activity inhibitor comprising, as an active ingredient, one or more plants selected from the group consisting of plants belonging to the genus Azalea, Viraceae, or a plant extract of those plants. 2. The calpain activity inhibitor according to claim 1, wherein the plant belonging to the genus Vaccinaceae is bilberry and the plant belonging to the genus Viraceae is a bamboo leaf. The calpain activity inhibitor of Claim 1 or 2 which uses the extract obtained by extracting the said solvent using ethanol, water-containing ethanol, or water as an active ingredient. It is an external preparation, The calpain activity inhibitor of any one of Claims 1-3.