JP3441395B2 - Endothelin converting enzyme inhibitor - Google Patents

Description

Description: TECHNICAL FIELD [0001] The present invention relates to an endothelin converting enzyme (hereinafter, referred to as “ECE”) inhibitor. [0002] Endothelin has a strong vasoconstrictor action and has been suggested to be a causative factor of pulmonary hypertension, ischemic heart disease, chronic renal failure and the like (Jpn. Pharmacol. 19).
92; 58; 279p etc.). Such endothelin is cleaved and produced from big endothelin (proendothelin) by ECE. Therefore, inhibition of ECE is considered to be effective for prevention, treatment, etc. of the above-mentioned diseases. For this reason, ECE inhibitors have been searched for, and for example, specific quinazoline derivatives (Japanese Patent Application Laid-Open No. 10-510834) are known. [0003] However, there has been a need for an ECE inhibitor which is effective, safe and has a wide range of applications. SUMMARY OF THE INVENTION The present inventors have found that, out of a wide range of plants, extracts of specific plants have a high ECE inhibitory effect. That is, the present invention relates to a product of Hawthorn, Cracked Beetle, Clove, Eijitsu, Asenyaku, Bodaiju, Licorice, Souhakuhi, Rosemary, Assamcha,
An ECE inhibitor comprising as an active ingredient an extract of one or more plants selected from the group consisting of birch, tormentilla, thyme, gentian, gennoshoko, linden, cassava, camellia, kawamogi, giant syrup, honeysuckle and mint. It is. BEST MODE FOR CARRYING OUT THE INVENTION The plants used in the present invention are Crataegus oxyacantha L., Warmomko (Sanguisorba officinalis L.) and Clove (Syzygi).
um aromaticum Merrill. et Perry, Rosa
multiflora Thunberg), Asenyaku (Uncaria gambir)
Roxburch, Bodaiju (Tilia platyphyllos Sco)
p.), licorice (Glycyrrhiza uralensis Fisher), sow oak (Morus alba Linne), rosemary (Rosmarinus)
officinalis L.), Assamcha (Thea sinensis Li)
nne var.), Birch (Betula platyphylla Sukatsche)
v var.), Tormentilla (Potentilla tormentilla Vul)
garis L.), thyme (Thymus vulgaris L.), gentian (Gentiana lutea L.), gennoshoko (Geranium)
thunbergii), carrot (Panax ginseng CA Maye)
r), Japanese linden (Cinchona succirubra Pavon), Japanese cypress (Swertia japonica (Schult.) Makino), Centella asiatica (Centellaasiatica L.), Artemisia (Artemisia)
capillaris Thunb.), Jio (Rehmannia glutinosa)
Libosch.), Honeysuckle (Lonicera japonica Thun)
b.) and mint (Mentha piperita L.). Among them, Warmoko has a haemostatic and platelet-increasing action; Coji, Eijitsu, Asenyaku, licorice, and Jiao have an inhibitory effect on testosterone 5α-reductase; Bodige has an anti-allergic effect; Rosemary, Gennoshoko and Chinoki have a superoxide-eliminating effect; Tea has a hyaluronidase-inactivating effect; birch and gentian have an anti-inflammatory effect; thyme and scorpion have a hair-growth and hair-growing effect; carrots and carrots have an androgen receptor binding inhibitory effect; It has been known. However, it has never been known that these plants have an ECE inhibitory action. [0008] The extract of the plant used in the present invention may comprise the whole plant or one or more of its leaves, petiole, stem, root and seed, dried or not dried, as it is or after grinding. Solvent extraction at room temperature or under heating, or extraction with an extraction device such as a Soxhlet extractor, or extraction with a supercritical gas such as carbon dioxide, or steam distillation, an extract obtained by a method such as pressing, Includes dilute or concentrated solutions, dry powders and the like. Water as the extraction solvent; alcohols such as methanol, ethanol, propanol and butanol;
Polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; halogenated hydrocarbons such as dichloromethane Hydrocarbons such as hexane, cyclohexane and petroleum ether;
Aromatic hydrocarbons such as toluene; polyethers such as polyethylene glycol; pyridines; and the like can be used alone or as a mixture of two or more kinds. In the extraction, a solvent is added to the whole plant of the above plant, etc.
Preferably 1 to 100 ° C, particularly preferably 3 to 70 ° C
, Preferably for 0.5 to 30 days, particularly preferably 1 to 30 days.
Perform for 15 days. A plant extract can be obtained by appropriately leaving the obtained extract at room temperature, filtering or the like. Such an extract may be subjected to liquid-liquid partitioning, removal of a solvable precipitate, or the like to remove inactive contaminants from the extract, and if necessary, may be deodorized or decolorized by a known method. Further, a fraction having high activity may be fractionated by gel filtration, chromatography, microfiltration or the like. The above plant extract can be used as it is as an ECE inhibitor, but may be formulated as appropriate. The content of the plant extract in the ECE inhibitor of the present invention is from 0.00001 to 20% by weight, particularly from 0.0001 to 20% by weight in terms of solid content, from the viewpoints of effects, mixability, feeling of use, and the like.
10% by weight is preferred. The ECE inhibitor of the present invention can be administered either externally or internally, but is preferably administered externally, especially in the form of a skin external preparation. The dosage form can be arbitrarily selected according to the purpose, and can be cream, ointment, emulsion, lotion, solution, gel, pack, powder, stick, and the like. Further, the ECE inhibitor of the present invention can be used as cosmetics, quasi-drugs, pharmaceuticals and the like. The ECE inhibitor of the present invention includes purified water, ethanol, oily substances, humectants, thickeners, and the like generally used in cosmetics, quasi-drugs, pharmaceuticals, etc. as long as the effects of the present invention are not impaired. Preservatives, emulsifiers, medicinal ingredients, powders, ultraviolet absorbers, pigments, fragrances, emulsion stabilizers, pH adjusters, and the like can be blended and manufactured according to a conventional method. Such ECE inhibitors include
It can be used for prevention and treatment of diseases such as pulmonary hypertension and ischemic heart disease. ECE includes endothelin 1, endothelin 2, endothelin 3 and the like, and the ECE inhibitor of the present invention is effective for any of them. EXAMPLES Preparation Example 1 Preparation of a Wild Hawthorn Extract A mixture of water and ethanol (50:50) was added to 10 g of the fruits of the hawthorn (Crataegus oxyacantha Linne).
0 ml was added, and the mixture was extracted at room temperature with occasional stirring for 24 hours. This was filtered and a mixture of water and ethanol (5
0:50) to make a total of 100 ml. Production Example 2 Preparation of Warmoko Extract Extract The roots and rhizomes of Warmoko (Sanguisorba officinalis Linne) are finely chopped, and 100 g of a mixed solution of water and ethanol (50:50) is added to 10 g of the root and rhizome at room temperature with occasional stirring. For 24 hours. This was filtered, 4 g of activated carbon was added to the filtrate, and the mixture was stirred at room temperature for 24 hours. This was filtered, and a mixture of water and ethanol (50:50) was added.
The whole was adjusted to 100 ml. Preparation Example 3 Preparation of Clove Extract A mixture of water and ethanol (50:50) was added to 10 g of buds of Clove (Syzygium aromaticum Merrill et Perry).
100 ml, add 24 hours at room temperature with occasional stirring.
After time extraction, the mixture was filtered. 100 ml of water is added to this and 40
The mixture was concentrated under reduced pressure at about 20 ° C. to about 20 ml. After performing this operation 5 times, water and ethanol were added to adjust the ethanol concentration to 50 v / v%, and the whole was adjusted to 100 ml. Production Example 4 Preparation of Age Extract An Age extract was prepared in the same manner as in Production Example 1, except that ground hazel (Rosa multiflora Thunberg) was used instead of the hawthorn. Preparation Example 5 Preparation of Asenyaku Extract In Preparation Example 1, a dried extract (Asenyaku) prepared by boiling the leaves and shoots of Uncaria ganbir Roxburgh was used in place of the fruit of Hawthorn. In the same manner as in Production Example 1, an Acacia catechu extract was prepared. Production Example 6 Preparation of Bodaiju Extract The flowers of Fudaidaiju (Tilia cordata Miller) are finely chopped, and 100 g of a mixed solution of water and propylene glycol (58:42) is added to 10 g of the flower and stirred at room temperature with occasional stirring. For 24 hours. This was filtered and a mixed solution of water and propylene glycol (58:42) was added to adjust the total volume to 100 ml. Preparation Example 7 Preparation of Licorice Extract Glycyrrhiza glabra Linne roots were cut into small pieces, and 5 g of the roots were immersed in 15 ml of a mixture of water and ethanol (5:95). This was filtered, and the obtained extract was concentrated to obtain a solid content of 325 mg. Ethyl acetate (10 ml) was added thereto, and the mixture was filtered again to remove insolubles, thereby obtaining a licorice extract. When this licorice extract was concentrated, its solid content was 56 mg. Preparation Example 8 Preparation of Saw Mulberry Extract The procedure of Preparation Example 1 was the same as that of Preparation Example 1, except that the fruits of the hawthorn were cut from the root bark of Mawwa (Morus alba Linne). An extract was prepared. Preparation Example 9 Preparation of rosemary extract The same procedure as in Preparation Example 1 was carried out except that the leaves and flowers of Mannenou (Rosmarinus officinalis Linne) were used in place of the fruits of the hawthorn. To prepare a rosemary extract. Preparation Example 10 Preparation of Assamcha Extract In Preparation Example 1, instead of the fruit of the hawthorn, the fruit of Assamcha (Thea sinensis Linne var. Assamica Pier) was used.
re) An assamcha extract was prepared in the same manner as in Production Example 1 except that tea (tea) made from leaves was used. Preparation Example 11 Preparation of Birch Extract Preparation Example 1 was the same as Preparation Example 1 except that the fruit of the hawthorn was replaced with a cut piece of bark and xylem of European birch (Betula pendula Roth.). A birch extract was similarly prepared. Preparation Example 12 Preparation of Tormentilla Extract In Preparation Example 1, the roots of tormentilla (Potentilla tormentilla Schrk) were used in place of the fruit of the hawthorn, and instead of a mixture of water and ethanol. Except for using water, a tormentilla extract was prepared in the same manner as in Production Example 1. Preparation Example 13 Preparation of Time Extract In Preparation Example 1, the same procedure as in Preparation Example 1 was carried out except that the above-ground portion of thymus serpyllum Linne was used instead of the fruit of Hawthorn. Time extract was prepared. Preparation Example 14 Preparation of Gentian Extract In Preparation Example 1, gentian (Gentiana lutea Linne) was used in the same manner as in Preparation Example 1 except that the root and rhizome of Gentiana lutea Linne were ground in place of the fruit of the hawthorn. An extract was prepared. Preparation Example 15 Preparation of Genoshoko Extract In Preparation Example 1, Geranium thunbergii Siebold et Zucca was used instead of the fruit of the hawthorn.
A genoshoco extract was prepared in the same manner as in Production Example 1, except that the above-ground portion of rini) was used. Preparation Example 16 Preparation of Carrot Extract Carrot extract was prepared in the same manner as in Preparation Example 1 except that roots of Panaxginseng CA Meyer were used in place of the fruits of Hawthorn. Prepared. Preparation Example 17 Preparation of Quina Extract In the same manner as in Preparation Example 1, except that the bark of Cinchona succirubra Pavon et Klotzch was used instead of the fruit of the hawthorn in Preparation Example 1, kina extract was used. Was prepared. Production Example 18 Preparation of Assembly Extract The assembly was performed in the same manner as in Production Example 1 except that the whole plant of Swertia japonica Makino was used in place of the fruit of the hawthorn. An extract was prepared. Preparation Example 19 Preparation of Centella asiatica extract The procedure of Preparation Example 1 was repeated, except that the leaves and stems of Centella asiatica Linne were used instead of the fruits of Hawthorn. A Centella extract was prepared. Preparation Example 20 Preparation of a Japanese Artemisia capillaris extract In Example 1, instead of the fruits of the hawthorn, cut flowers of Artemisia capillaris Thunberg were used, and 1,3 was used instead of ethanol.
-A mugwort extract was prepared in the same manner as in Production Example 1 except that -butanediol was used. Preparation Example 21 Preparation of Geo Extract Extract of Example 1 was replaced with Red Hawthorn (Rehmannia glutinosa Liboschitz var. Pu.
rpurea Makino)
Jio extract was prepared in the same manner as in Production Example 1. Preparation Example 22 Preparation of honeysuckle extract In Preparation Example 1, a flower of honeysuckle (Lonicera japonica Thunberg) was cut into pieces instead of the fruit of the hawthorn, and 1,3-butanediol was used instead of ethanol. Except for using, a honeysuckle extract was prepared in the same manner as in Production Example 1. Preparation Example 23 Preparation of Common Mentha Extract In the same manner as in the Preparation Example 1, except that the leaves of the common mint (Methapiperita Linne) were used in place of the fruits of the hawthorn extract, the same as in Preparation Example 1 was used. A mint extract was prepared. Test Example 1 (1) Preparation of ECE Human cultured vascular endothelial cells grown confluently on a flask coated with collagen (1) were washed with phosphate buffered saline (pH 7.4) and washed per 75 cm ² . 1m
L of 50 mM sodium phosphate buffer (pH 7.8)
Was added and the cells were scraped using a cell scraper.
Transfer the cell suspension to model GE 50 sonicator (serial 1
9214C) and crushed by ultrasonication at 10,000 g × 20
For 4 minutes at 4 ° C. 80,000 g of supernatant
Centrifuge at 4 ° C for 60 minutes, and precipitate 0.1% Trit.
25 mM sodium phosphate buffer containing on X-100
(PH 6.8) to give an ECE extract. (2) Enzyme reaction 0.1M sodium phosphate buffer (pH 6.8),
0.5 M NaCl, 2 μg of the ECE extract, and the above plant extract were mixed to a final concentration of 1% (v / v), and pre-incubated at 37 ° C. for 15 minutes. Then, 0.1 μM human big endothelin 1 was added and 10
After the reaction at 37 ° C. for 2 hours, 5 mM EDTA
Was added to stop the reaction. (3) Quantification of Endothelin Produced Endothelin in the reaction solution obtained above was quantified by a sandwich method using an enzyme immunoassay (endothelin-1 measurement kit: IBL). That is, first, 100 μL of the reaction solution after the completion of the above reaction and a standard solution of human endothelin 1 were added to a 96-well plate in which rabbit IgG against human endothelin 1 was immobilized, and reacted at 37 ° C. for 1 hour. Human endothelin 1 standard solution is 1% BSA, 0.0
Phosphate buffered saline containing 5% Tween 20 (pH 7.
Prepared by dilution in 4). The phosphate buffered saline (p
After washing the plate with H7.4), 0.3 μg of anti-endothelin-1 rabbit Fab ′ peroxidase-labeled antibody was added,
The reaction was performed at 37 ° C. for 30 minutes. After washing the plate with the phosphate buffered saline (pH 7.4), 100 μL of O-phenylenediamine (substrate) dissolved at 0.4 mg / mL in a buffer containing 0.03% hydrogen peroxide was added. Was added. After reacting for 15 minutes at room temperature in a dark place, 1N sulfuric acid was added for 1 minute.
The reaction was stopped by adding 00 μL. Then, using an ELISA plate reader (model 3550: Bio-Rad), 4
The absorbance at 90 nm was measured. (4) Calculation of ECE inhibitory activity The ECE inhibitory activity of each plant extract was calculated by the following equation, using the case where the above plant extract was not contained as a control. ECE inhibitory activity = {1− (absorbance when each plant extract is contained / absorbance of control)} × 100
(%). Table 1 shows the results. [Table 1] As is clear from Table 1, each plant extract exhibited excellent ECE inhibitory activity. Among them, the hawthorn, the scallop and the clove had an ECE inhibitory activity of 90% or more and were particularly excellent. The conversion of big endothelin into endothelin can be effectively suppressed by using the ECE inhibitor of the present invention.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 43/00 111 A61P 43/00 111 C12N 9/99 C12N 9/99 (72) Inventor Atsushi Ouchi Kaikaicho, Haga-gun, Tochigi Prefecture 2606 Akabane Kao Co., Ltd. (72) Inventor Kimihiko Hori 2606 Akabane, Kaigacho, Haga-gun, Tochigi Prefecture Kago Co., Ltd. (72) Inventor Hiroshi Kusunoki 2606, Kaigamachi, Akabane, Haga-gun, Tochigi Pref. ) References Nakajima Shigekat su et al, The Ginseng Radix Rubra on human vascular objective cells, American J. China Medicine, 1998, Vol. 26, No. 3-4, pp. 365-373 (58) Fields surveyed (Int. Cl. ⁷ , DB name) A61K 7/00 A61K 35/78 BIOSIS (STN) CA (STN) JICST file (JOIS) MEDLINE (STN) EMBASE (STN)

Claims (1)

(57) [Claims] [Claim 1] Wild hawthorn, Waremokou, Clover, Ages, Asenyaku, Bodaiju, Licorice, Sawhakuhi, Rosemary, Assamcha, Birch, Tormentilla, Thyme, Gentian, Gennoshoko, Japanese linden, Assemblage An endothelin converting enzyme inhibitor comprising, as an active ingredient, an extract of one or more plants selected from the group consisting of cinnamon, boxworm, sagebrush, scallop, honeysuckle and mint.