JP4992008B2 - Endothelin-1 production inhibitor - Google Patents

Description

The present invention relates et Ndoserin -1 production inhibitor.

  Endothelin-1 is a strong vasoconstrictor peptide produced by vascular endothelial cells and consists of 21 amino acids. Endothelin-1 is produced from big endothelin by the action of endothelin converting enzyme present in vascular endothelial cells. Endothelin-1 activates various intracellular signal transduction systems such as activation of phospholipase C and opening of calcium channels via the endothelin receptor of target cells. As a result, various effects such as regulation of hormone / neurotransmitter secretion and cell proliferation / differentiation are exhibited. In general, an increase in the concentration of endothelin-1 causes diseases such as hypertension and heart disease. Therefore, if endothelin-1 production can be suppressed, diseases such as hypertension can be treated or prevented.

  Endothelin-1 is also produced in various tissue cells other than blood vessels and plays various physiological roles. For example, when ultraviolet light hits the skin, endothelin converting enzyme works actively, big endothelin in keratinocytes is decomposed and endothelin-1 is produced, and endothelin-1 thus produced stimulates melanocytes, Melanin is actively synthesized to cause stains and buckwheat. Therefore, if the production of endothelin-1 can be suppressed, spots and buckwheat can be treated or prevented.

  There is a need in the market for drugs that treat or prevent hypertension, heart disease, spots, and freckles. In particular, hypertension is a serious disease that places a heavy burden on the cardiovascular system if left untreated for a long time, causes arteriosclerosis, and causes symptoms such as cerebral infarction and heart disease. Blood pressure is 140/90 mmHg or more. It is estimated that there are about 30 million patients with hypertension in Japan. One in four people and one in two over 50 years old are considered hypertensive. Is anxious.

In order to meet such market demand, endothelin-1 production inhibitors have been studied. Conventionally studied as endothelin-1 production inhibitors are mainly chemical synthetic products, but considering safety, natural products having similar effects are desired. As natural products, polyphenols (piceatannol, delphinidin, etc.) (see Non-Patent Document 1) and soy isoflavone (Genistein) (see Non-Patent Document 2) contained in red wine extract have been used as vascular endothelial cell endothelin-1 Although it has been studied as a production inhibitor of sucrose, it cannot be said that it sufficiently meets market demands in terms of safety and cost. For example, some isoflavones exhibit an estrogen-like action, and there are concerns about the adverse effects of overdose. Thus, pharmaceutical, of a safe and inexpensive endothelin-1 production inhibitor which can be used in such cosmetics is still a need.

Prior art document information related to this application includes the following.
Nature (UK), 2001, 414, 863-864 Atherosclerosis, (Ireland), 2002, 163, 339-347

  The subject of this invention is providing the endothelin-1 production inhibitor derived from a natural product.

The present invention includes the following inventions.
(1) An endothelin-1 production inhibitor containing luteolin and / or its glycoside.
(2) An endothelin-1 production inhibitor containing a plant extract containing luteolin and / or its glycoside.
(3) An endothelin-1 production inhibitor containing an extract of Passiflora edulis (also called passion fruit).
(4) The endothelin-1 production inhibitor according to the above (3), wherein the extract of damselfly is a peel extract or a pulp extract.

  The present invention provides a natural product-derived endothelin-1 production inhibitor with high safety.

  Hereinafter, although the said this invention is demonstrated in detail, the scope of the present invention is not restrained by these explanations, and it can implement suitably in the range which does not impair the meaning of this invention except the following illustration.

  As a result of earnest search for endothelin-1 production inhibitors derived from natural products, the present inventors have the effect that luteolin and / or its glycosides act on vascular endothelial cells to suppress endothelin-1 production. I found.

  That is, the present invention relates to an endothelin-1 production inhibitor containing luteolin and / or a glycoside thereof. Luteolin (3 ', 4', 5, 7-tetrahydroxyflavone) is a kind of flavonoid and is a natural product contained in celery, peppers, spring chrysanthemum, chamomile, shiso and the like. Moreover, various glycosides exist in nature, for example, 6-C-fucosyl luteolin and 6-C-quinovosyl luteolin have been isolated from Kuda monodiatom (Phytochemistry, 30, 3486, 1991). Conventionally, it is not known that luteolin or its glycoside has an effect of suppressing the production of endothelin-1.

  In the present invention, luteolin may be a commercially available product, but may be isolated from various plants and used. The luteolin glycoside that can be used in the present invention may be a glycoside of luteolin and any sugar (eg, glucose, fucose, galactose, 6-deoxyglucose), and the binding position of the sugar is, for example, 6 of the luteolin molecule. -C, 8-C, 7-O, or 4'-O, and a preferred luteolin glycoside is luteolin-6-C-glucoside.

  The endothelin-1 production inhibitor containing luteolin and / or its glycoside according to the present invention may be luteolin and / or its glycoside itself, or a commonly used solid or liquid carrier, emulsified dispersion It may be formulated into a dosage form such as a tablet, powder, emulsion, capsule, etc. Examples of the carrier include water, gelatin, starch, magnesium stearate, lactose, and vegetable oil. The endothelin-1 production inhibitor may contain further components.

  The content of luteolin and / or its glycoside in the endothelin-1 production inhibitor of the present invention can be appropriately changed depending on the administration purpose, administration route, dosage form, etc. ~ 20% by weight.

The present invention further relates to an endothelin-1 production inhibitor containing a plant extract containing luteolin and / or its glycoside. Since the plant extract is high and inexpensive safety, medicines, are particularly suitable for use in such cosmetics. Here, the plant extract is a liquid usually extracted by dissolving components contained in the whole plant or parts such as roots, stems, leaves, flowers, fruits, etc. in a solvent such as water or ethanol, and drying appropriately. Or a solid thing. In the present invention, an extract of any plant can be used as long as it contains luteolin and / or its glycoside. For example, in addition to damselfly, Lamiaceae plants such as blue dissociation, red dissociation, egoma, Extracts such as celery family plants such as celery and ashitaba, vegetables such as celery, lettuce, spring chrysanthemum and bell pepper, soybean leaves, peanut seed coat, ginkgo biloba, honeysuckle, western primrose, chamomile and peppermint can be used. Of these, an extract of damselfly is preferred.

  Furthermore, this invention relates to the endothelin-1 production inhibitor containing the extract of a damselfly. As the extract of cereals, fruit skin, fruit, fruit, leaf, stem or root extract is preferable, and fruit skin extract or pulp extract is particularly preferable.

  Kudamono Passiflora is a vine perennial tropical fruit tree native to South America and is now widely distributed in tropical and subtropical regions around the world and is cultivated industrially in Australia, Southeast Asia, Taiwan and other countries. In Japan, it is the most produced in Okinawa, but it is also cultivated in the Amami and Ogasawara Islands. There are no particular limitations on the variety and production area of the damselfly used in the present invention.

  For example, the citrus peel extract that can be used in the present invention is crushed with a mixer or the like after finely crushing the peel of the pearl fly diatom, and then insoluble solvent is removed by centrifugation or filtration. It can be obtained as a powder by drying under reduced pressure. In the present invention, the “fruit skin” means a skin portion outside the fruit, but generally said pulp portion may be attached. The extraction solvent may be any solvent, and for example, one or more of methyl alcohol, ethyl alcohol, ethyl acetate, other organic solvents, and water can be used.

  For example, the fruit extract of Kudamono Passiflora that can be used in the present invention is stirred with water, and the insoluble matter is removed by centrifugation or filtration, and the supernatant or filtrate is separated from an ultrafiltration membrane having a molecular weight cut off of 10,000. The solution that has been passed through and recovered can be obtained as a powder by drying under reduced pressure. In the present invention, “fruit pulp” means a semi-transparent jelly-like portion of a fruit, but generally referred to skin and seed portions may be attached. The extraction solvent may be any solvent, and for example, one or more of methyl alcohol, ethyl alcohol, ethyl acetate, other organic solvents, and water can be used.

  The endothelin-1 production inhibitor containing a plant extract containing luteolin and / or its glycoside of the present invention, or the endothelin-1 production inhibitor containing an extract of damselfly is an extract itself. Alternatively, it may be formulated into a dosage form such as a tablet, powder, emulsion, capsule or the like by a commonly used solid or liquid carrier, emulsifying dispersant and the like. Examples of the carrier include water, gelatin, starch, magnesium stearate, lactose, and vegetable oil. The endothelin-1 production inhibitor may contain further components.

  The content of the plant extract in the endothelin-1 production inhibitor of the present invention can be appropriately changed depending on the purpose of administration, the administration route, the dosage form, and the like. Is 1.0 to 80% by weight with respect to the total weight of the preparation, and when it contains the extract of citrus fruit juice, the content is 1.0 to 90% by weight with respect to the total weight of the preparation.

  The endothelin-1 production inhibitor of this invention can be used as a pharmaceutical.

  The usage form of the endothelin-1 production inhibitor of the present invention may be any form depending on the purpose of use, for example, oral administration is possible. In addition, cosmetics containing an endothelin-1 production inhibitor are typically applied directly to the skin.

  The dose of the endothelin-1 production inhibitor of the present invention is not particularly limited, and is appropriately determined according to, for example, the type of illness, the patient's age, sex, weight, symptom level, or administration method. be able to.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Test Example 1 Endothelin-1 Production Inhibitory Activity of Luteolin and Luteolin Glycoside Porcine Aortic Endothelial Cells (passage number 1, 25 cm ^{2 in} culture flask, purchased from Cell Systems Corporation) in CS-C medium (D -MEM medium and Ham F12 medium are mixed in an equal ratio of 1: 1 to a medium supplemented with 10% fetal bovine serum, 15 mM HEPES, acidic FGF, and heparin (purchased from Cell Systems Corporation), and collagen is dispersed. It was added to one coated 24-well plate (1 cm ² cm ² ) and cultured at 37 ° C. for 4 days in the presence of 5% CO ₂ . The medium in each well of the plate is then discarded and 1 ml of Krebs-Ringer bicarbonate (KRB) buffer (NaCl 129 mM, NaHCO ₃ 5 mM, KCl 4.8 mM, KH ₂ PO ₄ 1.2 mM, CaCl ₂ 1.0 mM, Each well was washed twice with MgSO ₄ 1.2 mM, glucose 2.8 mM, bovine serum albumin 0.1%, HEPES 10 mM, PH 7.4). Then, 40 μl sample in 360 μl KRB buffer (63-500 μM luteolin in 10% DMSO (dimethylsulfoxide) aqueous solution, 250-1000 μM luteolin glycoside in 10% DMSO aqueous solution, or 10% DMSO aqueous solution as a control) 400 μl of the solution (reaction solution) added with was added to each well, left in the presence of 5% CO ₂ at 37 ° C. for 0 to 8 hours, and the concentration of endothelin-1 in the solution was measured. The results are shown in Table 1 and FIG. Table 1 shows the amount of endothelin-1 released into the solution during 3 hours, the amount of endothelin-1 (control) at the time of addition of 10% DMSO aqueous solution (final concentration of DMSO in the reaction solution) of 100%. As a comparison value (average value of n = 4). FIG. 1 shows the endothelin-1 concentration (average value of n = 3) in the solution at each time point after sample addition. Luteolin and luteolin glycoside (luteoline-6-C-glucoside) were found to have an endothelin-1 production inhibitory effect.

  Next, we examined the effects of citrus peel and flesh extract on cell growth using MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) kit (Roche) As a result, it was confirmed that the concentration range in Table 1 had no effect on cell proliferation.

Test Example 2 Production Method of Endothelin-1 Production Inhibitor Consisting of Extract of Kudamono Passiflora Skin Extract Peeled peel (150 g) from 3 Kudamono passiflora fruits (purchased in Naha city) with a mixer, and then 200 ml of methanol In addition, the mixture was sufficiently stirred (electromagnetic stirrer, 400 rpm, 60 minutes). Subsequently, filtration with a filter paper (Advantech Toyo filter paper, No. 2, diameter 30 cm) was performed to remove insoluble matters, and the filtrate was recovered. The filtrate was concentrated with a rotary evaporator and further dried under reduced pressure using a vacuum pump to obtain 3.6 g of a purple powder.

Test Example 3 Production Method of Endothelin-1 Production Inhibitor Composed of Kudamono Passiflora Fruit Extract 110 ml of jelly-like flesh (including seeds) was collected from 3 Kudamono passiflora fruits, and the same amount of distilled water was added thereto. Stir well (electromagnetic stirrer, 400 rpm, 60 minutes). Subsequently, centrifugation was performed at 15,000 rpm for 20 minutes to precipitate insoluble matter, and the supernatant was collected. This solution was passed through an ultrafiltration membrane YM-10 (molecular weight cut off 10,000, Millipore), and the filtrate was concentrated with a centrifugal evaporator and dried under reduced pressure with a vacuum pump to obtain a light orange powder of 16 g. Got.

Test Example 4 Endothelin-1 Production Inhibitory Activity of Kudamonodiaceutum Skin Extract and Flesh Extract As in Test Example 1, porcine aortic endothelial cells cultured in 24-well plates were washed twice with KRB buffer, and then 360 μl of KRB buffer. 400 μl of solution (reaction solution) with 40 μl of sample (2.5-40 mg / ml aqueous solution of citrus peel extract, 0.75-7.5 mg / ml aqueous solution of citrus fruit extract, or distilled water as a control) Was added to each hole and allowed to stand at 37 ° C. for 0-8 hours in the presence of 5% CO ₂ . The number of cells at the time of sample addition is 4.0 × 10 ⁵ per well. Thereafter, 200 μl of the supernatant was collected, and the contaminated cells were removed by centrifugation. The amount of endothelin-1 in the supernatant was measured by enzyme immunoassay using an endothelin-1 ELISA system (Amersham Biosciences). The results are shown in Table 2 and FIG. Table 2 shows the amount of endothelin-1 released into the solution during 3 hours as a value (average value of n = 4) compared with the amount of endothelin-1 at the time of adding distilled water (control) as 100%. . FIG. 2 shows the endothelin-1 concentration (average value of n = 3) in the solution at each time point after sample addition. An inhibitory effect on endothelin-1 production was observed in the citrus peel extract and the citrus peel extract.

  Next, as in Test Example 1, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) kit (Roche) was used to extract citrus peel extract against cell proliferation and When the influence of the pulp extract was examined, it was confirmed that there was no influence on cell proliferation in the concentration range shown in Table 2.

It is a figure which shows the density | concentration change of the endothelin-1 in the porcine aortic endothelial cell in a KRB buffer at the time of adding luteolin. It is a figure which shows the density | concentration change of the endothelin-1 in the porcine aortic endothelial cell in a KRB buffer solution at the time of adding a damselfly fruit peel extract.

Claims (4)

Endothelin-1 production inhibitor containing luteolin and / or its glycoside (however, it is an endothelin-1 production inhibitor for the treatment or prevention of heart disease, hypertension, blemishes or buckwheat, and whitening cosmetics) Except for endothelin-1 production inhibitors) . Endothelin-1 production inhibitor containing a plant extract containing luteolin and / or its glycoside (however, an endothelin-1 production inhibitor for the treatment or prevention of heart disease, hypertension, blemishes or buckwheat, and , Except for endothelin-1 production inhibitor which is a cosmetic for whitening) . Endothelin-1 production inhibitor containing an extract of damselfly (however, endothelin-1 production inhibitor for treatment or prevention of heart disease, hypertension, stains or buckwheat, and endothelin-1 which is a whitening cosmetic product) Excluding production inhibitors) .   The endothelin-1 production inhibitor according to claim 3, wherein the extract of damselfly is a peel extract or a pulp extract.

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