JP5745751B2 - Composition for improving dry syndrome - Google Patents

Description

  The present invention relates to a composition for improving dry syndrome, which contains a nitric oxide (NO) -producing substance as an active ingredient.

  Dry syndrome (dry syndrome) is a dry phenomenon that occurs in various parts of the body due to problems in the living environment. Typical diseases include dry mouth (xerostomia), dry eye (dry eye), dry skin (dry skin) and the like. In dry mice, salivary secretion is reduced, and the mouth is dry and sticky, causing discomfort such as caries, periodontal disease, bad breath, unclear taste, and sleep disorders. In addition, dry eyes are accompanied by symptoms such as dry eyes, pain in the eyes, redness of the eyes, and eye discomfort. Also, with dry skin, the skin moisture and sebum amount are insufficient, and the skin becomes dry. It is said that there are an estimated 8 million patients in dry eye and dry mouse. Since these symptoms cause a significant reduction in the quality of life (QOL) of the consumer, it is desirable to be improved by appropriate treatment.

  On the other hand, nitric oxide (NO) functions as a physiologically functional substance in the living body. The nitric oxide (NO) is produced when citrulline is produced from arginine by NO synthase. Conventionally, research on the relationship between the physiological function of nitric oxide and the development of pathological conditions has been actively conducted, and by now, by increasing nitric oxide in the whole body or a predetermined body part, angina pectoris or myocardium It has been reported that diseases such as ischemic heart disease such as infarction, cerebral infarction, arteriosclerotic arterial occlusion, various infectious diseases, impotence and the like are improved or treated (see Patent Document 1 below).

  However, there was no report that the dry syndrome was improved by increasing nitric oxide.

JP 7-324039 A

  An object of the present invention is to provide a composition for improving dry syndrome, which has an effect of improving symptoms of dry syndrome such as dry mouth, dry eye, and dry skin.

  As a result of diligent research to achieve the above object, the present inventors have found that a nitric oxide (NO) -producing substance has an action effect of enhancing the water permeation function of living cells, and to complete the present invention. It came.

That is, the present invention provides a composition for improving dry syndrome having the following constitution.
[1] A composition for improving dry syndrome, comprising a nitric oxide (NO) -producing substance as an active ingredient.
[2] The composition for improving dry syndrome according to the above [1], which has the effect of promoting saliva secretion, tear secretion, or increasing skin moisture.
[3] The composition for improving dry syndrome according to [1], which has an effect of promoting saliva secretion.
[4] The composition for improving dry syndrome according to any one of [1] to [3], wherein the nitric oxide (NO) -producing substance is citrulline, arginine, or argininosuccinic acid.

  According to the composition for improving dry syndrome of the present invention, the nitric oxide (NO) -producing substance as an active ingredient enhances the water permeation function of living cells. The effect of promoting or increasing the moisture of the skin is brought about, and thereby symptoms of dry syndrome such as dry mouth, dry eye, and dry skin can be improved. In particular, by using citrulline, arginine, or argininosuccinic acid as a nitric oxide-producing substance, the above effect can be enhanced.

It is the microscope picture of the dome formation of MDCK I monolayer cell, and its schematic diagram. It is a graph which shows the result of the dome formation number (number of domes / field) at the time of NO stimulation. 6 is a chart showing the results of the number of domes / field when NO stimulation is inhibited by NO scavengers. It is a graph which shows the result of an AQP3 / GAPDH gene expression ratio after 20-hour NO stimulation of MDCK I cells. It is a graph which shows the change of the amount of saliva after ingesting citrulline with various intakes of 250 mg (A) 500 mg (B) and 2000 mg (C). It is a graph which shows the result of having represented the change of the amount of saliva in various intake amount over the elapsed time.

  In the composition for improving dry syndrome of the present invention, a nitric oxide (NO) -producing substance is used as an active ingredient for improving the dry syndrome.

  In the present invention, the nitric oxide (NO) -producing substance is an effect of promoting the production of nitric oxide in vivo by, for example, ingestion, instillation, or application to the skin as an external preparation. Means a substance having

  Preferred examples of the substance that produces nitric oxide include citrulline, arginine, and argininosuccinic acid. That is, in vivo generation of nitric oxide occurs through a reaction cycle represented by the following conceptual formula, so if an intermediate substance in the reaction cycle is ingested, nitric oxide is generated through the reaction cycle. .

  In particular, citrulline is preferable because it reaches the target body part without being decomposed, metabolized or excreted by the liver. Citrulline has long been used as an OTC drug overseas, and is a type of amino acid that was approved as a food in Japan in 2007, and is abundant in vines such as watermelon. Wild watermelons that grow naturally in intense sunlight and extremely dry and harsh environments contain a particularly large amount of citrulline, which is thought to play a major role in surviving dry environments.

  Examples of the nitric oxide production promoting substance include tiger cane, japonica, serpentine, malt, mulberry bark, glutinous rice, yam, float, huangyong, yongkou, oysters described in JP-A-7-324039. One or more herbal medicines selected from beneficial herbs, Aoki incense, carrots and yellow sesame or extracts thereof can also be used.

  In the present invention, the dry syndrome is a dry phenomenon that occurs in various parts of the body due to problems in the living environment, and typical examples of the dry syndrome include dry mouth (dry mouth) and dry eye (dry eye). , Dry skin (dry skin). The composition for improving dry syndrome of the present invention has an effect of promoting saliva secretion, lacrimal fluid secretion, and increasing the moisture of the skin. Therefore, dry mouth (xerostomia), dry eye (dryness) It can be preferably applied to both eyes) and dry skin (dry skin).

  The composition for improving dry syndrome of the present invention is a tablet, granule, powder, powder, and the like, by adding a pharmaceutically acceptable base material or carrier to the nitric oxide production promoting substance as described above, if necessary. In addition to oral preparations such as liquids, powders, granules, capsules, jelly-forms, etc., ophthalmic external preparations such as eye drops, ointments, creams, gels, packs, lotions, cosmetics, etc. It can be commercialized as an external preparation for skin. In addition, the composition for improving dry syndrome of the present invention can also be ingested by being blended with food for specified health use, dietary supplement, functional food and the like. Examples of such foods include confectionery such as chocolate, biscuits, gum, candy, cookies, gummies, tablet confectionery, etc .; cereals; powdered drinks, soft drinks, milk drinks, nutritional drinks, carbonated drinks, jelly drinks, etc. Beverages; frozen desserts such as ice cream and sorbet. Furthermore, noodles such as buckwheat, pasta, udon, and somen can be preferably exemplified. Moreover, in the case of foods for specified health use, dietary supplements, and the like, they may be in the form of powders, granules, capsules, syrups, tablets, sugar-coated tablets and the like.

  In the present invention, the composition for improving dry syndrome may be taken orally and allowed to act from within the body. Alternatively, it may be applied externally from outside the body. In the following, the mode of use will be further described separately when applied to dry mouth, dry eye and dry skin.

-Dry mouse The dry syndrome improving effect of the present invention can be obtained by orally ingesting the dry syndrome improving composition of the present invention or by directly acting on the saliva secretion pathway so as to remain in the oral cavity for a certain period of time. . For example, in the examples described later, it is shown that the effect of promoting continuous saliva secretion can be obtained by orally ingesting citrulline as a nitric oxide-producing substance. In order to improve dry mice, the amount of intake when taken orally, for example, using citrulline as a nitric oxide-producing substance, is preferably about 200 to 3000 mg in terms of citrulline per adult day. Of course, an oral preparation such as a candy, troche, chewable tablet, mouthwash and the like may be directly acted on the salivary secretion pathway so as to remain in the oral cavity for a certain period of time. In the case of candy, troches, chewable tablets, etc., when citrulline is used as the nitric oxide producing substance, for example, citrulline is preferably contained in the composition in an amount of 0.5 to 100% by mass. Moreover, in the case of mouthwash etc., it is preferable to make it contain about 0.01-20 mass% in the composition, and to apply.

-Dry eye By ingesting the composition for improving dry syndrome of the present invention or administering it as an eye drop, secretion of tears is promoted and an effect of improving dry eye can be obtained. In order to improve dry eye, the amount of intake when taken orally, for example, using citrulline as a nitric oxide-producing substance is preferably about 200 to 3000 mg in terms of citrulline per adult day. In addition, for example, citrulline is used as a nitric oxide-producing substance, and the amount applied when it is administered as an external preparation for eye drops such as eye drops is about 1 to 6 times per use, and 0.005 to 10 mg per use. It is preferable that it is a grade.

-Dry skin By ingesting the composition for improving dry syndrome of the present invention or applying it to the skin as an external preparation for skin, the moisture of the skin can be increased and the effect of improving dry skin can be obtained. In order to improve dry skin, the amount of intake when taken orally using, for example, citrulline as a nitric oxide-producing substance is preferably about 200 to 3000 mg per day for adults. In addition, an external preparation for skin such as an ointment, cream, gel, pack, lotion, cosmetics, etc. may be allowed to act directly on the skin so that it stays at the application site for a certain period of time. For example, when citrulline is used as the nitric oxide-producing substance, the effective application amount is preferably about 2.5 to 30 mg / cm ² per unit area.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

<Test Example 1>
The effect of NO stimulation by nitric oxide-producing substances on the water permeation function of living cells was examined using cultured cells. MDCK I cells were used as cultured cells for that purpose. MDCK I (Madin-Darby Canine Kidney) cells are canine kidney-derived cells, and the cells form a monolayer sheet on the plate. In addition, it was known that when cells were stimulated, some of the cells swelled in an arch shape, forming a “dome” in which salt and water inside and outside the cell flowed and accumulated in the gap between the culture plate and the monolayer cell ( Fig. 1 shows a micrograph of the dome formation and a conceptual diagram.) Therefore, if such dome formation was promoted, it was thought that the enhancement of the water permeation function of cells could be evaluated. Specifically, the test was conducted as follows.

[Cell culture]
MDCK I cells derived from canine kidney purchased from DS Pharma Biomedical Co., Ltd. were cultured in a MEM medium (manufactured by GIBCO) containing 10% FBS in a 5% CO ₂ environment at 37 ° C. according to a conventional method. In addition, 100 U / ml penicillin and 100 mg / ml streptomycin were added to the medium.

[Dome formation test by NO stimulation]
The MDCK I cells were seeded on a 24-well culture plate at a cell density of 2.5 × 10 ⁵ cells / well, and it was confirmed that the cells reached confluence. As a nitric oxide producing substance for NO stimulation, NOC18 (manufactured by Dojin Chemical Laboratories Co., Ltd.), which is a NO producing agent, was used. This NOC18 was dissolved in 10 mM NaOH and added to the cells that reached the confluence so that the final concentrations were 0, 0.05, 0.1, 0.2, and 0.5 mM. After incubating for 20 hours in a 5% CO ₂ environment at 37 ° C, the number of domes / number of domes / field). FIG. 2 shows the result in a graph.

[NO stimulation inhibition by NO scavenger]
The effect of erasing NO with a NO erasing agent was investigated. Specifically, a reagent that selectively reacts with NO to generate NO ₂ and eliminates NO, Carboxy-PTIO (2- (4-Carboxyphenyl) -4, 4, 5, 5-tetramethylimidazoline-1- oxyl-3-oxide) (manufactured by Dojindo Laboratories Co., Ltd.) is added to the cells that have reached the confluence at final concentrations of 0.1, 0.2, 0.4 mM, and then NOC18 is added to a final concentration of 0.2 mM. Incubated for 20 hours at 37 ° C. in 5% CO ₂ environment. The number of dome formations was counted in the same manner as described above. FIG. 3 shows the result in a graph.

[result]
By adding NOC18, which is a NO producing agent, to MDCK I cells, an increase in the number of dome formation was observed in a concentration-dependent manner (FIG. 2). As a result of erasing NO by NOC18 with Carboxy-PTIO, dome formation was suppressed depending on the Carboxy-PTIO concentration (Fig. 2). Therefore, it was revealed that the dome formation of MDCK I monolayer cells was enhanced by NO stimulation.

<Test Example 2>
It was confirmed that the dome formation of MDCK I monolayer cells was enhanced by NO stimulation. Therefore, by examining the gene expression level of aquaporin (AQP), which is known to be greatly involved in the water permeation function (water permeability) of living cells in animals and plants, what mechanism is this dome formation by NO stimulation? We examined what happens. Specifically, the test was conducted as follows.

[Cell culture]
MDCK I cells were cultured in the same manner as in Test Example 1 above.

[Quantitative PCR]
Semi-confluent MDCK I cells were detached with trypsin EDTA, seeded in a 96-well plate at 8 × 10 ⁴ cells / well, confirmed to have reached confluence, replaced with fresh medium, and NOC18 dissolved in 1N NaOH was replaced The medium was added to contain 0.5 mM. After 20 hours from the addition, the cells were collected, and from cell lysis to quantitative PCR were performed using TaqMan Gene Expression Cells-to-CT Kit (Ambion) according to a conventional method. At that time, PCR primers for quantification of the AQP 3 gene and PCR primers for quantification of the GAPDH gene for quantification control were those shown in Table 1 below.

[result]
As shown in FIG. 4, the AQP 3 / GAPDH gene expression ratio after MDCK I cells were stimulated with NO for 20 hours was 4.7 times that before stimulation. Therefore, the expression of aquaporin (AQP 3) is involved in the dome formation of MDCK I monolayer cells by NO stimulation, and is caused by increased water permeability through the expression of aquaporin (AQP 3) It was inferred.

<Test Example 3>
According to Test Examples 1 and 2 above, it was presumed that the water permeation function of living cells was enhanced by NO stimulation with a nitric oxide-producing substance, and this was tried to be verified by a human test. Specifically, it was examined whether or not the amount of saliva increases in the oral cavity by ingesting citrulline, which is known to have NO production effects in the body.

[Test method]
As test solutions, 250, 500, and 2000 mg / 30 ml of citrulline solutions in which commercially available citrulline (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water were prepared. The subjects collected humans with a low saliva volume (less than 1 mL by the following method) in a preliminary test. And on the day before the test, I asked them to avoid intense exercise and avoid eating and drinking after 21:00. On the day of the test, the test was conducted with a rest time of at least 5 minutes after sitting.

  First, saliva before sample intake was collected by chewing salivette cotton (SARSTEDT) at an interval of 1 time / second for 60 seconds. Sampling was performed three times at 5-minute intervals, and the average value was taken as the amount of saliva before ingestion. As the test food, 30 mL of water or 30 mL of citrulline solution was ingested, and the test was performed without disclosing the water or the concentration of citrulline solution to the subject. The amount of saliva 30, 60, 90, 120 minutes after ingestion was collected using salivet cotton as described above. The collected salivet cotton was centrifuged at 3000 rpm for 5 minutes, and the amount of saliva was measured using a graduated glass capillary micropipette (DRUMMND). The amount of saliva was expressed as a relative value with the amount of saliva before ingestion (average of 3 times) being 100.

[result]
FIG. 5 is a graph showing changes in the amount of saliva divided into cases where citrulline was ingested at various intakes of 250 mg (A), 500 mg (B) and 2000 mg (C). As shown in FIG. 5, by taking 250 mg (A), 500 mg (B), or 2000 mg (C) of citrulline, the relative amount of saliva at each time compared to the water intake of the control at each time. It can be seen that is rising. In addition, citrulline has a relatively high saliva relative to water even after 90 minutes of ingestion, and it does not promote transient saliva secretion during ingestion but has a continuous salivary improvement effect. It was.

  FIG. 6 is a graph showing changes in the amount of saliva with various intakes, averaged over the elapsed time. According to FIG. 6, it can be seen that the relative saliva amount is increased depending on the citrulline concentration as a whole after citrulline intake.

  From the above, it was confirmed that citrulline, which is a nitric oxide-producing substance, has an effect of promoting salivary secretion. Moreover, since this action effect is not a transient effect but a sustained effect, it has been clarified that this can effectively improve the symptoms of dry mice.

SEQ ID NO: 1: AQP 3 gene forward primer SEQ ID NO: 2: AQP 3 gene reverse primer SEQ ID NO: 3: GAPDH gene forward primer SEQ ID NO: 4: GAPDH gene reverse primer

Claims (1)

An oral composition for promoting saliva secretion , comprising citrulline, arginine, or argininosuccinic acid as an active ingredient.