JP2013253049A - Epithelial sodium channel expression inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an epithelial sodium channel expression inhibitor having an excellent epithelial sodium channel expression inhibition effect.SOLUTION: An epithelial sodium channel expression inhibitor includes sesame peptide as an active ingredient. Further, a renal sodium resorption inhibitor includes sesame peptide as an active ingredient.

Description

  The present invention relates to an epithelial sodium channel expression inhibitor.

Epithelial Na ⁺ channel (hereinafter referred to as “ENaC” in this specification) is an ion channel localized in the epithelial cell membrane. It selectively permeates sodium ions and participates in body fluid regulation in vivo. To do. ENaC is composed of α, β, and γ subunits. In particular, expression of α subunit (hereinafter, also referred to as “αENaC” in the present specification) is considered to be important for ENaC activity.

  The human renal cortical collecting duct (distal renal tubule) is composed of a connecting tubule and a cortical, outer medullary layer, and inner medullary collecting duct. Among these, ENaC is expressed in the tubular membrane. ENaC's renal physiological function is known to precisely control the amount of sodium in the body by reabsorbing sodium in the renal cortical collecting duct, which is very useful for regulating body fluid volume, plasma osmotic pressure, blood pressure, etc. It has an important role. In particular, ENaC is responsible for sodium influx into the renal cortical collecting duct cells.

  On the other hand, aldosterone, a kind of mineral corticoid, synthesized and secreted in the adrenal cortex globular layer, has a function of controlling the balance of sodium ions and potassium ions in the blood. Specifically, aldosterone is activated by the loss of fluid, and the sodium pump is activated by significantly increasing the ENaC number and activating the sodium pump, so that sodium ions taken into the tubular cells via ENaC Pumped out with a sodium pump, enhances reabsorption of sodium into renal cortical collecting duct cells and decreases sodium excretion in the urine. The influx of sodium into the renal cortical collecting duct cells causes the tubular lumen side to become electrically negative and promotes potassium secretion via the potassium channel. Therefore, when the expression of ENaC is excessively increased and the reabsorption of renal sodium is promoted, the renal function may be deteriorated (see, for example, Non-Patent Documents 1 and 2).

Other than kidney function deterioration, overactive cystitis, dry eye, dry mouth, xeroderma, etc. are known as diseases caused by excessive expression of ENaC (hyperfunction mutation) (for example, , Patent Documents 1 to 5, Non-Patent Document 3).
From such a background, it can be expected that by suppressing the expression of ENaC, an effect of preventing / ameliorating the symptoms developed through ENaC as described above can be expected.

Special table 2010-526118 gazette Special table 2009-520729 JP 2005-530692 A JP 2006-232684 A JP 2010-59210 A

F. R. Perez et al., Hypertension, vol. 53, p. 1000-1007, 2009 T. Nakayama et al., Am. J. Physiol. Renal. Physiol., Vol. 296, p. F317-F327, 2009 Masayuki Takeda et al., “Role of epithelial sodium channel (ENaC) in the mechanism of overactive bladder development”, Autonomic Nerve, Japan Autonomic Neurology Society, 43 (4), p.309-314, 2006-08-15

  An object of the present invention is to provide an ENaC expression inhibitor having an excellent ENaC expression suppression effect. The present invention also includes an inhibitor of renal sodium reabsorption, a renal function improving agent, an overactive cystitis improving agent, an ocular dryness improving agent, an xerostomia improving agent, and an xeroderma ameliorating agent that suppresses ENaC expression The issue is to provide.

  In view of the above problems, the present inventors have conducted intensive studies. As a result, sesame peptide has an excellent inhibitory effect on ENaC expression and is useful for improving renal function by suppressing reabsorption of sodium into the kidney, active cystitis, dry eye, dry mouth Has been found to be useful in the prevention and improvement of psoriasis and xeroderma. The present invention has been completed based on these findings.

That is, the present invention relates to an epithelial sodium channel expression inhibitor containing a sesame peptide as an active ingredient.
The present invention also includes a renal sodium reabsorption inhibitor, a renal function improving agent, an overactive cystitis improving agent, a dry eye (dry eye) improving agent, a dry mouth improving agent, which contains a sesame peptide as an active ingredient, And an agent for improving xeroderma.

  ADVANTAGE OF THE INVENTION According to this invention, the ENaC expression inhibitor which has the outstanding ENaC expression suppression effect can be provided. Further, according to the present invention, ENaC expression is suppressed, renal sodium reabsorption inhibitor, renal function improving agent, overactive cystitis improving agent, dry eye ameliorating agent, xerostomia ameliorating agent, and xeroderma amelioration An agent can be provided.

  Epithelial sodium channel expression inhibitor, renal sodium reabsorption inhibitor, renal function improving agent, overactive cystitis improving agent, dry eye (dry eye) improving agent, dry mouth disease improving agent, and xeroderma The improving agent contains sesame peptide as an active ingredient.

The sesame peptide used in the present invention is a sesame seed proteolysate obtained by treating sesame seeds with a proteolytic enzyme.
Sesame seeds are rich in protein, and sesame peptides can be obtained by enzymatic degradation of this protein. Sesame peptide is known to have a blood pressure lowering action and an antioxidant action (refer to Japanese Patent No. 2820868, etc.), and since it is manufactured using sesame as a raw material, it is highly safe, so it is incorporated into various supplements and the like. Has been.

The sesame peptide used in the present invention is widely available to the public. For example, it may be extracted from sesame seeds or commercially available ones may be used.
Examples of commercially available products include sesame peptide KM-20 manufactured by KISCO Corporation, sesame peptide manufactured by Kishimoto Sangyo Co., Ltd., and the like.

When sesame peptide is extracted from sesame seeds, the seeds of sesame genus (sesame seed, scientific name: Sesamum indicum ), scabaceae (linseed, scientific name: Linum usitatissimum , also known as flax) Seeds) can be used. In the present invention, sesame seeds collected from these natural products as raw sesame seeds, as well as processed sesame seeds and defatted sesame seeds produced in the sesame oil production process may be used. Among these, it is preferable to use defatted sesame as a raw material.
In the present invention, these are collectively referred to as sesame seeds.

The sesame peptide used in the present invention is obtained by extracting sesame protein from the above sesame seeds and decomposing the extracted sesame protein by an enzymatic reaction.
The method for extracting sesame protein from sesame seeds is not particularly limited. For example, Journal of Food Science, Vol. 47, pp. 457-460 (1982). According to this method, sesame protein can be obtained as a precipitate by isoelectric point precipitation or the like after degreasing raw sesame seeds with hexane, dissolving and extracting the protein from the obtained defatted sesame with an alkaline solution.

  The enzyme used for the decomposition | disassembly of sesame protein is not specifically limited, A normal proteolytic enzyme can be used. From the viewpoint of safety, it is preferable to use a proteolytic enzyme for food. For example, Alcalase (registered trademark: manufactured by Novo Industry Japan Co., Ltd.), Samoaze (trade name: manufactured by Daiwa Kasei Co., Ltd.), Pro Leather (registered) Trademark: manufactured by Amano Pharmaceutical Co., Ltd.), protin FA (trade name: manufactured by Daiwa Kasei Co., Ltd.) and the like can be used.

  What is necessary is just to set suitably about the conditions of the enzyme reaction at the time of proteolysis in consideration of the optimal reaction temperature and optimal reaction pH of the proteolytic enzyme to be used. The same applies to the amount of enzyme added and the reaction time. From the viewpoint of the recovery rate of the protein degradation product, etc., it is preferably a condition under which a peptide bond degradation rate of 3% or more is obtained, and more preferably a condition under which a peptide bond degradation rate of 15% or more is obtained.

After the enzyme reaction, the target protein degradation product is recovered from the enzyme reaction solution, but this method is not particularly limited. For example, as a method of removing undegraded sesame protein and used proteolytic enzyme, a method of filtering after heat treatment or isoelectric point precipitation treatment, a method of ultrafiltration, or the like can be applied.
The obtained protein degradation product may be used in the present invention as it is, or may be diluted or concentrated. In addition, it is possible to use a protein degradation product that is appropriately dried by a method such as freeze-drying to obtain a paste or powder.

As shown in Examples described later, sesame peptide can suppress the expression level of ENaC gene. Therefore, the sesame peptide can be used for suppressing biological functions performed via ENaC, or for preventing or ameliorating diseases or symptoms caused by overexpression of the ENaC gene.
An example of the suppression of biological function performed via ENaC includes suppression of sodium reabsorption action on renal cortical collecting duct cells performed via ENaC. In addition, diseases and symptoms caused by overexpression of NaC gene include exacerbation of renal function due to excessive enhancement of ENaC expression and promotion of reabsorption of renal sodium, and overactivity caused by overexpression of ENaC gene. Cystitis, dry eye (dry eye), xerostomia and xeroderma (Reference, JP 2010-526118, JP 2009-520729, JP 2005-530692, JP 2006 No. 232684, JP 2010-59210 A, Masayuki Takeda et al., “Role of Epithelial Sodium Channel (ENaC) in Overactive Bladder Pathogenesis”, Autonomic Nerve 43 (4), p.309-314, 2006- 08-15, etc.). That is, sesame peptide can be used for the suppression of these effects or the prevention and improvement of symptoms.
The use may be a therapeutic use (ie medical practice) or a non-therapeutic use (non-medical practice). Moreover, the subject of the use can be a human, a non-human animal, or a specimen derived therefrom.

Epithelial sodium channel expression inhibitor containing sesame peptide as an active ingredient, renal sodium reabsorption inhibitor and renal function improving agent, overactive cystitis improving agent, dry eye disease improving agent, dry mouth disease improving agent, and dry skin The disease-improving agent is one of the specific modes of use described above, and can be applied to both therapeutic uses (medical uses) and non-therapeutic uses (non-medical uses). The usage forms include all forms that can be applied to human, animal skin, nails, mucous membranes, hair and the like.
Specific examples include use as pharmaceuticals, quasi-drugs, cosmetics, etc., in the case of cosmetics, quasi-drugs, and pharmaceuticals, in the form of external skin preparations, and in the case of pharmaceuticals, in the dosage form can do. Moreover, these agents can also be mix | blended as an active ingredient in various food-drinks, pet food, etc.

Epithelial sodium channel expression inhibitor, renal sodium reabsorption inhibitor, renal function improving agent, overactive cystitis improving agent, dry eye ameliorating agent, dry mouth ameliorating agent, and xeroderma ameliorating agent These agents are collectively referred to as “the agent of the present invention”) may contain only the sesame peptide that is the active ingredient, or contain other ingredients within a range that does not affect the effect. It may be a thing. Other components can be appropriately selected according to the form and use of the agent.
When other ingredients are added to the agent, the content of the sesame peptide as an active ingredient is not particularly limited, but the sesame peptide is preferably contained in the agent in an amount of 0.001% by mass or more, 0.01% by mass or more More preferably, it is more preferably 0.1% by mass or more. Moreover, it is preferable that 40 mass% or less of sesame peptide is contained in an agent, It is more preferable that 20 mass% or less is contained, It is further more preferable that 10 mass% or less is contained.

When the agent of the present invention is used as a pharmaceutical in the form to be administered to humans or animals, for example, oral solid preparations such as tablets, coated tablets, granules, powders, capsules, oral liquids such as oral liquids and syrups It can be in the form of parenteral preparations such as preparations, injections, external preparations, suppositories, and transdermal absorption agents.
In preparing these preparations, excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, Diluents and the like can be appropriately blended.
When used in the above dosage form, the amount of sesame peptide in the agent is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and further preferably 1% by mass or more. Moreover, 40 mass% or less is preferable, as for the upper limit of a compounding quantity, 10 mass% or less is more preferable, and 4 mass% or less is further more preferable.

When the agent of the present invention is used in the form of an external preparation for skin, the “external preparation for skin” includes those applied to the skin as skin cosmetics, external pharmaceuticals, quasi-drugs for external use, and the like. The mold can take a wide variety of forms such as an aqueous solution system, a solubilization system, an emulsification system, a powder system, a gel system, an ointment system, a cream, a water-oil two-layer system, and a water-oil-powder three-layer system. More specifically, facial cleanser, lotion, lotion, milky lotion, cream, gel, essence (beauty serum), pack, mask, massage agent, foundation, lipstick, bath preparation, shampoo, hair conditioner, hair tonic, ointment, Examples include pastes, sheet-like products (absorbent articles such as sanitary products, wiping wipes, wet tissues, etc.).
In addition, in making these dosage forms, various oil agents, surfactants, gelling agents, preservatives, antioxidants, solvents, alcohol, water, chelating agents, thickeners, ultraviolet absorbers, emulsion stabilizers, pH A regulator, a pigment, a fragrance, and the like can be appropriately blended.
0.001 mass% or more is preferable, as for the compounding quantity of the sesame peptide in a skin external preparation, 0.1 mass% or more is more preferable, and 1 mass% or more is further more preferable. Moreover, 40 mass% or less is preferable, as for the upper limit of a compounding quantity, 10 mass% or less is more preferable, and 4 mass% or less is further more preferable.

When formulating the agent of the present invention as an active ingredient in foods and drinks, pet foods, etc., in addition to general foods and drinks, it develops due to excessive expression of ENaC, and the concept of prevention or improvement of symptoms effective in suppressing ENaC expression is therapeutic If necessary, it can be added to and blended with functional foods and beverages such as beauty foods, sick foods, nutritional functional foods, and foods for specified health use.
There is no particular limitation on the type of food and drink, for example, beverages such as fruit juice beverages, milk beverages, tea-based beverages, confectionery such as candy, drop, jelly, cookies, chocolate, cakes, yogurt, gum, seasonings, Examples include cooking oil, dairy products, breads, noodles, and processed rice. Moreover, it is good also as beauty foods, health foods, etc., such as a tablet (tablet), a capsule, a granule, and a syrup.
In these foods and drinks, food ingredients such as sweeteners, colorants, antioxidants, vitamins, fragrances, minerals, etc., proteins, lipids, carbohydrates, carbohydrates, dietary fibers, etc. should be appropriately combined. Can do.
When mix | blending with food-drinks, pet food, etc., 0.001 mass% or more is preferable, 0.001 mass% or more is preferable, 0.01 mass% or more is more preferable, and 0.1 mass% or more is further more preferable. Moreover, 20 mass% or less is preferable, as for the upper limit of a compounding quantity, 5 mass% or less is more preferable, and 1 mass% or less is further more preferable.

When the agent of the present invention is used in the form of pharmaceuticals or the like, or when the agent of the present invention is blended with food or drink, etc., the amount used depends on the condition, weight, sex, age, or other factors of the person to be applied. Can vary. The administration or intake of sesame peptide per day for an adult (body weight 60 kg) is preferably 0.01 mg or more, more preferably 0.1 mg or more, further preferably 1 mg or more, and 1 g or less. It is preferable that it is 0.5 g or less, more preferably 0.3 g or less.
The agent of the present invention is administered three times a day, twice a day, once a day, once every two days, once every three days, once a week, or at any period and interval, or Can be ingested.
When the agent of the present invention is used in the form of a skin external preparation, the amount of the skin external preparation used varies depending on the content of the active ingredient in the agent, but is 0.003 g or more and 0.5 g or less per 1 cm ^{2 of} the skin surface. It is preferable that

  In relation to the above-described embodiment, the present invention further discloses the following method.

<1> A method for suppressing the expression of epithelial sodium channels (excluding medical practice), characterized by orally ingesting a food or drink containing sesame peptide.
<2> A method for suppressing reabsorption of kidney sodium (excluding medical practice), characterized by orally ingesting a food or drink containing sesame peptide.
<3> A method for improving renal function (excluding medical practice), characterized in that food or drink containing sesame peptide is taken orally.
<4> A method for preventing or improving overactive cystitis, wherein a food or drink containing sesame peptide is taken orally (excluding medical practice).
<5> A method for preventing or improving dry eye disease (excluding medical practice), characterized by orally ingesting a food or drink containing sesame peptide.
<6> A method for preventing or improving xerostomia (excluding medical practice), characterized by orally ingesting a food or drink containing sesame peptide.
<7> A method for preventing and improving xerosis (excluding medical practice), characterized by orally ingesting a food or drink containing sesame peptide.
<8> A method for preventing and improving xeroderma (excluding medical practice), characterized by applying a cosmetic containing sesame peptide to the skin.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Example 1. Preparation of Sesame Peptide Sesame peptide (KM-20) manufactured by KISCO Corporation was used and dissolved in DMSO (dimethyl sulfoxide) to prepare a sesame peptide sample. The sample solution is added to the following 2. The sesame peptide was added to the system at a concentration of 10 μg / mL.

2. Evaluation of ENaC expression inhibitory action As a culture solution, 10% fetal bovine serum (FBS, manufactured by Invitrogen) and 1% penicillin streptomycin in a growth factor-supplemented medium (D-MEM / Ham's F-12 (1: 1, manufactured by Invitrogen)) A solution (to which Invitrogen) was added) was used. Mouse well cortical collecting duct cells (trade name: M-1, manufactured by DS Pharma Biomedical) 24-well cell culture multiwell plate (flat bottom, manufactured by Falcon) so that the number of cells is 1 × 10 ⁴ cells / well And cultured for 3 days in the culture solution. Thereafter, the cells were cultured for 1 day in D-MEM / Ham's F-12 (FBS (-)) to which no FBS was added, and the medium was changed.

  It acts on GR and on mineralocorticoid receptor (MR) on M-1 cells cultured as described above and reported to undergo signal transduction via a glucocorticoid receptor (GR) -mediated pathway. Like aldosterone, dexamethasone (50 nM) having ENaC expression promoting action and the sesame peptide sample prepared above were added to a final concentration of 10 μg / mL and cultured for 24 hours. As a control, the same test was performed except that the same amount of dimethyl sulfoxide (DMSO) was added.

The cultured cells were collected, and total RNA was extracted using RNeasy (registered trademark) mini kit (Qiagen). Furthermore, using the obtained RNA as a template, Omniscript (registered trademark) Reverse Transcriptase (Invitrogen) (RNase-free water 1.6 μL, 5 × First strant bffer 4 μL, 0.1 M dTT 2 μL, 10 mM sNTP Mix 1 μL, 0.5 μg / μL oligo (dT) 0.4 μL, 200 unit / μL M-MLVRT 1 μL) was mixed and subjected to reverse transcription reaction to synthesize a cDNA sample. Using the synthesized cDNA sample as a template, the gene expression level of ENaC α subunit (αENaC) was analyzed using TaqMan Fast Universal PCR Master Mix (trade name, manufactured by Applied Biosystems) (reagent: TaqMan Fast Universal PCR Master). Mix, apparatus: ABIPRISM7500Sequence Detector (trade name, manufactured by Applied Biosystems) was used. The expression level of the αENaC gene was corrected using 36B4 (housekeeping gene) as an internal standard, and the value was relative to the expression level of 36B4. As PCR primers and TaqMan probes, TaqMan (registered trademark) gene expression assays (Product Number: Mm00803386_m1 (αENaC), Mm99999223_gH (36B4)) were used. The obtained analysis results are shown by using an average value ± standard deviation, and a significant difference test with respect to the control group was performed by Dunnett.
The results are shown in Table 1.

As is clear from the results in Table 1, the expression level of αENaC gene was significantly suppressed in the system to which sesame peptide was added. In other words, the sesame peptide exhibited an αENaC gene expression-inhibiting action and suppressed ENaC expression.
Therefore, the epithelial sodium channel (ENaC) expression inhibitor of the present invention containing sesame peptide as an active ingredient has an excellent ENaC expression suppression effect. In addition, by using sesame peptide as an active ingredient, reabsorption of sodium into renal cortical collecting duct cells via ENaC is suppressed, and further, reabsorption of renal sodium is promoted by excessive expression of ENaC. Since the deterioration of the renal function that occurs can be improved, it is possible to provide a renal sodium reabsorption inhibitor and a renal function improver. Further, by using the sesame peptide as an active ingredient, preventive / ameliorating agents for various symptoms caused by ENaC overexpression, that is, an overactive cystitis ameliorating agent, a dry eye ameliorating agent, an xerostomia ameliorating agent and It is also possible to provide an agent for improving xeroderma.

Claims (7)

  An epithelial sodium channel expression inhibitor comprising sesame peptide as an active ingredient.   A renal sodium reabsorption inhibitor comprising sesame peptide as an active ingredient.   A renal function improving agent comprising sesame peptide as an active ingredient.   An overactive cystitis improving agent comprising sesame peptide as an active ingredient.   A dry eye amelioration agent comprising sesame peptide as an active ingredient.   An agent for improving xerostomia containing sesame peptide as an active ingredient.   A dry skin ameliorating agent comprising sesame peptide as an active ingredient.