JP6486738B2 - VEGFR3 expression reduction improving agent - Google Patents

Description

  The present invention comprises a red ganoderma extract and / or a black ganoderma extract extracted with carbon dioxide in a temperature range of 20 to 150 ° C. and a pressure range of 1 to 80 MPa. It relates to an improving agent.

  Capillary lymphatic vessels in the dermis of the skin collect tissue fluids and proteins that have leaked into peripheral tissues, and fat absorbed from the small intestine, and drain waste products to maintain circulation of tissue fluids in the body. ing. Such a circulation of tissue fluid can be said to be indispensable for maintaining the homeostasis of the body fluid and the physiological activity of the tissue.

  Capillary lymph vessels consist of a single layer of lymphatic endothelial cells. Lymphatic endothelial cells include VEGFR3, which is a transmembrane receptor tyrosine kinase that is specifically expressed, and vascular endothelial growth factor-C (VEGF-C) has been identified as a factor that activates VEGFR3. As a result of this signal transmission via VEGF-C / VEGFR3, formation of new lymphatic vessels is promoted after lymphatic endothelial cells proliferate and migrate (lymphangiogenesis).

  The VEGF family, including VEGF-C, is composed of multiple factors. While VEGF-A works to promote angiogenesis, it has a function of deteriorating the function of lymphatic vessels, and VEGF-B is supposed to work specifically in blood vessels. It is known that VEGF-C acts in a lymphatic vessel-specific manner and is a major factor that promotes lymphangiogenesis.

  Swelling is a symptom that is caused when the function of lymphatic vessels is reduced.

  Swelling is caused by the fact that tissue fluid in the body is not properly drained, and excess water and proteins accumulate in the interstitial tissue and swell, resulting in daily routines such as sitting and standing for a long time. It is likely to occur naturally. If the swelling is left for a long time, it is not only cosmetically unfavorable, but it may cause various adverse effects on the human body such as accumulation of cellulite that leads to obesity.

  Conventionally, as a method for relieving swelling, it is well known that the flow of lymph fluid is promoted by physical force from outside the body, such as drug application or massage, and the circulation of tissue fluid in the body is activated. Examples of drugs having an effect of improving swelling include cosmetics and foods containing plant extracts and the like. For example, a swelling improving agent containing salmon as an active ingredient (Patent Document 1), and swelling containing α-glycosylated rutin as an active ingredient. Techniques such as food for improvement (Patent Document 2) have been reported.

  On the other hand, edema occurs in skin with reduced VEGF-C (Non-patent Document 1), and when the binding of VEGF-C to VEGFR3 is inhibited using a VEGFR3 neutralizing antibody, the number of lymphatic vessels is significantly reduced. However, it has been reported that edema occurs (Non-patent Document 2). It is also known that inflammation can cause lymphedema (Patent Document 3). Conversely, it has also been shown that inflammation and edema due to ultraviolet light are recovered by improving the activity of VEGFR3 by administration of VEGF-C (Non-patent Documents 1 and 2). Thus, when VEGF-C / VEGFR3 signaling is reduced by various stimuli, swelling occurs, and it is considered that improving such reduction in signaling leads to improvement of swelling.

  As a technique for improving the decrease in VEGF-C / VEGFR3 signal transduction, one or more kinds of drugs selected from the group consisting of Kyonin extract, carrot extract, valerian extract, hawthorn extract, merirot extract, nettle extract, and Iris root extract are used. There are reports of a VEGF-C production promoter characterized by containing (Patent Document 4), a swelling improving agent characterized by containing S-lactoylglutathione and / or a salt thereof (Patent Document 5), etc. .

  On the other hand, for red ganoderma extract and black ganoderma extract, biological gene damage inhibitors (Patent Document 6), thrombosis improving agents (Patent Document 7), whitening agents (Patent Document 8), hypertension inhibitors ( A technique such as Patent Document 9) is disclosed. Moreover, about the red ganoderma extract and the black ganoderma extract extracted with the carbon dioxide in the temperature range of 20-150 degreeC and the pressure range of 1-80 Mpa, whitening agent (patent document 10), the undifferentiation of a stem cell Techniques such as a maintenance agent and a growth promoter (Patent Document 11), a hair growth promoter (Patent Document 12), and a skin wrinkle formation preventing / improving agent (Patent Document 13) are disclosed. However, no reports have been made on the effect of improving the reduction in VEGFR3 expression and the effect of swelling on the red ganoderma extract and the black ganoderma extract.

Patent 4052750 JP2007-130020 WO2007 / 013603 Patent 5105890 Patent 5384285 JP 2002-322081 A Patent 4644936 Patent 4808580 JP2008-255041 Patent 3742084 JP2011-211956A JP2013-71927A JP2014-43413

J Invest Dermatol. (2009), Volume 129, pp 1292-1298. J Invest Dermatol. , (2006), Volume 126, pp920-922.

  In daily life, the skin is always exposed to external stimuli such as ultraviolet rays, drying and temperature changes. When such a stimulus damages the lymphatic vessel, VEGF-C / VEGFR3 signaling is inhibited and the function of the lymphatic vessel is reduced, the collection of tissue fluid in the body is delayed, and it is expected to cause new swelling symptoms.

  However, massages, swelling-promoting agents, and swelling-improving foods, etc., activate the water circulation to eliminate swelling, and do not directly affect the deterioration of lymphatic vessels that cause swelling. There was a problem that only an effect could be obtained.

  Also, normalization of VEGF-C / VEGFR3 signaling is important for improving lymphatic dysfunction, and a technique for improving swelling by promoting VEGF-C production has been reported. In the state where VEGFR3 expression is reduced, the effect is not sufficiently exhibited.

  This invention makes it a subject to provide the composition which has the outstanding effect which improves a VEGFR3 expression fall. It is thought that by improving the decrease in VEGFR3 expression, the lymphatic vessel function can be activated and swelling can be improved.

  As a result of intensive studies to solve this problem, the present inventors have found that red ganoderma extract and black extracted with carbon dioxide in a temperature range of 20 to 150 ° C. and a pressure range of 1 to 80 MPa. It was discovered that an excellent swell improvement effect can be obtained with Ganoderma lucidum extract, and the present invention has been completed.

  That is, the present invention contains a red ganoderma extract and / or a black ganoderma extract extracted with carbon dioxide in a temperature range of 20 to 150 ° C. and a pressure range of 1 to 80 MPa. It is an expression reduction improving agent.

  By using the red ganoderma extract and / or black ganoderma extract extracted with carbon dioxide in the temperature range of 20 to 150 ° C. and the pressure range of 1 to 80 MPa of the present invention, damage is caused by stimulation from the outside world. Decreased expression of VEGFR3 on the received lymphatic endothelial cell membrane can be improved, and swelling caused by no inflammation is also improved by activating lymphatic function.

  The red ganoderma and black ganoderma used in the present invention are basidiomycetes used in the herbal medicine “ganoderma” and belong to the family Ganodermaaceae and Ganoderma. As for mushrooms belonging to the genus Amanenchu, there are old Chinese pharmacy books such as “Honcho Tsuname” and “Shinnohonsyo”, red ganoderma (reishi), black ganoderma (black turf), purple ganoderma (purple shiba), It is described that blue ganoderma (blue turf), yellow ganoderma (yellow turf) and white ganoderma (white turf) exist. Kazuno Ganoderma is also a type of red ganoderma. The scientific names of Red Ganoderma and Ganoderma Ganoderma are (Ganoderma lucidum), and the scientific names of Black Ganoderma (G.atrum, G.japonicum, G.sinense). In general, there are a lot of red ganoderma, staghorn ganoderma and black ganoderma. Those widely distributed in the Chinese and Japanese markets can be used, or native products and cultivated products may be used. A mycelium culture can also be used. These may be used as they are or may be dried or pulverized.

  The extraction of the red ganoderma extract and the black ganoderma extract in the present invention can be performed according to a known extraction method using carbon dioxide in a supercritical state or a state in the vicinity thereof.

  The extraction conditions in the present invention are a temperature of about 20 to 150 ° C. and a pressure of about 1 to 80 MPa, preferably a temperature of about 30 to 60 ° C. and a pressure of about 10 to 50 MPa. Can do. The extraction time and the supply amount of carbon dioxide can be appropriately selected according to the purpose.

  Furthermore, an organic solvent can also be used as a cosolvent (entrainer) in the present invention. Examples of the cosolvent include ethanol and acetone. Of these, ethanol is preferable from the viewpoint of safety.

  Extraction can be performed, for example, by continuously blowing carbon dioxide under the above extraction conditions. Next, the carbon dioxide fluid containing the extract is introduced into a separation tank and separated by a commonly used method, for example, a method of lowering pressure or a method of changing temperature. At this time, the separation tank can be filled with an adsorbent that can adsorb the extracted solute, a medium (solvent, base) that can be dissolved or dispersed, and the like. Means can be adopted. The separated carbon dioxide can be transported to the liquefaction tank and reused.

  The extract may be used as it is, or may be used after treatment such as concentration, dilution, filtration, deodorization, and drying as necessary. Furthermore, the active ingredient may be concentrated or isolated by performing column purification or the like.

  According to the extraction method of the present invention, triterpenes such as ganoderic acid contained in red ganoderma can be extracted effectively. This effect is particularly remarkable when an entrainer is used.

  The content of the red ganoderma extract and the black ganoderma extract extracted with carbon dioxide in the temperature range of 20 to 150 ° C. and the pressure range of 1 to 80 MPa used in the present invention is as follows. The total amount of red ganoderma extract and black ganoderma extract is 0.001% by weight or more, preferably 0.01 to 5% by weight in terms of solid matter. The content ratio when the red ganoderma extract and the black ganoderma extract are used in combination is not particularly limited, but the black ganoderma extract has a weight ratio of 0.33 to 3 with respect to the red ganoderma extract 1. It is preferable because the improvement of expression and swelling of VEGFR3 is remarkable. More preferably, the black ganoderma extract is 1 in weight ratio to the red ganoderma extract 1. The addition method may be added in advance or may be added during production, and may be appropriately selected in consideration of workability.

  The swelling agent of the present invention can be used for cosmetics, quasi-drugs, and pharmaceuticals, and as a dosage form, application as a skin external preparation is effective.

  In the case of an external preparation for skin, in addition to the above-mentioned components, aqueous components, powders, oils, moisturizers, alcohols, pH adjusters other than the above components as long as the effects of the present invention are not impaired. In addition, preservatives, pigments, thickeners, fragrances, and the like can be blended as appropriate, and can be produced according to conventional methods. Specific examples of the form of the external preparation for skin include skin lotion, emulsion, cream, gel, ointment, bath preparation, gel, paste, essence, pack and the like.

  In order to describe the present invention in detail, examples of production, formulation and experimental examples of the extract used in the present invention will be given as examples, but the present invention is not limited thereto.

Production Example 1 Supercritical extract of red ganoderma 1 kg of ganoderma (red ganoderma) pulverized material is charged into an extraction tank with an internal volume of 5 L, and supercritical carbon dioxide (pressure 25 MPa, temperature 60 ° C., carbon dioxide supply amount) 15 m ³ ) was supplied for about 4.5 hours and led to a separation tank (temperature 40 ° C., pressure 4 MPa) connected to the extraction tank to separate carbon dioxide and the extract, thereby obtaining 10.0 g of the extract.

Production Example 2 Supercritical Extract of Black Ganoderma Red Ganoderma was replaced with Black Ganoderma in Production Example 1, and treated in the same manner as in Production Example 1 to obtain 10.8 g of extract.

Formulation Example 1 Cream Ingredient Content (wt%)
1. Red Ganoderma Supercritical Extract (Production Example 1) 0.05
2. Supercritical extract of Black Reishi (Production Example 2) 0.05
3. Squalane 5.5
4). Olive oil 3.0
5. Stearic acid 2.0
6). Beeswax 2.0
7). Octyldodecyl myristate 3.5
8). Polyoxyethylene cetyl ether (20E.O.) 3.0
9. Behenyl alcohol 1.5
10. Glycerol monostearate2.5
11. Fragrance 0.1
12.1,3-Butylene glycol 8.5
13. Methyl paraoxybenzoate 0.25
14 [Manufacturing method] Components 1 to 10 are heated and dissolved in purified water and mixed, and the mixture is kept at 70 ° C to obtain an oil phase. Ingredients 12-14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase was added to the oil phase to emulsify, and the mixture was cooled while stirring. The component 11 was added at 45 ° C, and the mixture was further cooled to 30 ° C to obtain a cream.

Comparative Example 1 Conventional Cream A conventional cream obtained by replacing the supercritical extract of red ganoderma and black ganoderma with squalane in Formulation Example 1.

Formulation Example 2 Cream Ingredient Content (wt%)
1. Red Ganoderma Supercritical Extract (Production Example 1) 0.05
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11.1,3-butylene glycol 8.5
12 Methyl paraoxybenzoate 0.25
13. [Manufacturing method] Components 1 to 9 are heated and dissolved and mixed with purified water to 100. The oil phase is maintained at 70 ° C. Ingredients 11-13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase was added to the oil phase to emulsify, and the mixture was cooled while stirring. The component 10 was added at 45 ° C., and further cooled to 30 ° C. to obtain a cream.

Formulation Example 3 Ointment Component Content (% by weight)
1. Red Ganoderma supercritical extract (Production Example 1) 0.005
2. Black reishi supercritical extract (Production Example 2) 0.015
3. Polyoxyethylene cetyl ether (30E.O.) 2.0
4). Glycerol monostearate 10.0
5. Liquid paraffin 5.0
6). Cetanol 6.0
7). Methyl paraoxybenzoate 0.1
8). Propylene glycol 10.0
9. [Production method] Components 1-6 were heated and dissolved and mixed with purified water to 100. The oil phase was maintained at 70 ° C. Ingredients 7-9 were dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. An aqueous phase was added to the oil phase to emulsify, and the mixture was cooled to 30 ° C. with stirring to obtain an ointment.

Formulation Example 4 Ointment Component Content (% by weight)
1. Supercritical extract of Black Reishi (Production Example 2) 0.04
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glycerol monostearate 10.0
4). Liquid paraffin 5.0
5. Cetanol 6.0
6). Methyl paraoxybenzoate 0.1
7). Propylene glycol 10.0
8). [Production method] Components 1-5 were heated and dissolved and mixed with purified water to 100. The oil phase was maintained at 70 ° C. Ingredients 6 to 8 were dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. An aqueous phase was added to the oil phase to emulsify, and the mixture was cooled to 30 ° C. with stirring to obtain an ointment.

Formulation Example 5 Gel Agent Component Content (% by weight)
1. Red Ganoderma supercritical extract (Production Example 1) 0.75
2. Supercritical extract of Black Reishi (Production Example 2) 0.25
3. Ethanol 5.0
4). Methyl paraoxybenzoate 0.1
5. Polyoxyethylene hydrogenated castor oil (60 EO) 0.1
6.1,3-Butylene glycol 5.0
7). Glycerin 5.0
8). Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Production method] Ingredients 1 to 5 and 6 to 11 are uniformly dissolved in purified water to 100, and both are mixed to obtain a product.

  Hereinafter, in order to explain the present invention effectively, an experimental example of the present invention is given as Example 3, but the present invention is not limited to this.

Experimental Example 1 Expression Change of VEGFR3 in LYVE-1 and PROX-1 Positive Endothelial Cells (HDMEC) In this study, as an example of external stimulation, inflammatory cytokine IL-1α is a specific marker for lymphatic vessels. -1 and PROX-1 in endothelial cells were examined for effects on VEGFR3 mRNA expression.
HDEC was seeded at 1 × 10 ⁵ in a 60 mm dish, and when it became confluent, 10 ng / ml IL-1α was added, and at the same time, supercritical extracts of red ganoderma and black ganoderma were added. After culturing, total RNA was extracted using RNAiso plus (TAKARA). Measurement of mRNA expression level was performed by real-time RT-PCR method based on total RNA extracted from cells. SYBR Select Master Mix (Life Technologies) was used for the real-time RT-PCR method. That is, 500 ng of total RNA was subjected to a reverse transcription reaction, followed by a PCR reaction (95 ° C .: 15 seconds, 60 ° C .: 60 seconds, 40 cycles). For other operations, the expression level of mRNA was determined as a ratio with respect to the expression level of GAPDH mRNA as an internal standard in accordance with a predetermined method. The primers used for measuring the expression level of each gene are as follows.

Primer set GGTCATCATGCTGTCTCTCTTCT (SEQ ID NO: 1) for VEGFR3
CAGGTAGCCCGTCTTGATGTC (SEQ ID NO: 2)
GAPDH primer set TGCACCACCAACTGCCTTAGC (SEQ ID NO: 3)
TCTTCTGGGTGGCAGTGATG (SEQ ID NO: 4)

  The obtained results are shown in Table 1. When IL-1α was added to HDMEC, the expression level of VEGFR3 mRNA decreased. When the supercritical extract of red ganoderma or black ganoderma was added in addition to IL-1α, the expression level of VEGFR3 mRNA changed in the recovery direction. When the supercritical extracts of red ganoderma and black ganoderma were added at the same time in addition to IL-1α, the expression level of VEGFR3 mRNA was remarkably recovered as compared with the addition alone. Therefore, it was shown that each of the supercritical extracts of red ganoderma or black ganoderma has an effect of improving the reduction of VEGFR3, which causes swelling due to decreased lymphatic function. In addition, it was shown that the supercritical extracts of red ganoderma and black ganoderma have the effect of more effectively improving the reduction of VEGFR3 when used in combination.

Experiment 2 Usage test
The cream of the formulation example 1 and the comparative example 1 was prepared, and the effect at the time of actually using was examined. Applying the cream of Formulation Example 1 to the right calf, and the cream of Comparative Example 1 to the left calf twice daily in the morning and evening for 15 female subjects (20-50s) who are usually suffering from swelling. And conducted a use test for one month. In the evaluation method, the left and right calf maximum perimeters in the morning and evening were measured one month after use, and the difference between the maximum calf perimeters in the morning and evening was taken as the amount of swelling. The average value was calculated | required and the swelling amount after using the comparative example 1 and the prescription example 1 for 1 month was compared. At the same time, a usability questionnaire was conducted to evaluate the impact on swelling.

  The obtained results are shown in Tables 2 and 3. When the formulation example 1 is used, it can be seen that the amount of swelling is clearly smaller than that of the comparative example 1, and swelling is reduced. In addition, from the usability questionnaire, it was confirmed that the feeling of swelling improvement due to the application of Formulation Example 1 was high. Therefore, the expression of VEGFR3 characterized by containing the red ganoderma extract and / or black ganoderma extract extracted with carbon dioxide in the temperature range of 20 to 150 ° C. and the pressure range of 1 to 80 MPa according to the present invention. It has been clarified that the reduction-improving agent exhibits an excellent swell improvement effect in a human use test. During the test period, there were no skin problems and no safety problems. The same test was conducted on the cream of Formulation Example 2, the ointment of Formulation Examples 3 and 4, and the gel preparation of Formulation Example 5, and a remarkable swelling improvement effect was recognized.

  Lymph damaged by stimulation from the outside by using a red ganoderma extract and / or a black ganoderma extract extracted with carbon dioxide in a temperature range of 20 to 150 ° C. and a pressure range of 1 to 80 MPa. The decrease in the expression of VEGFR3 on the vascular endothelial cell membrane can be improved. ADVANTAGE OF THE INVENTION By this invention, the agent which prevents and improves the swelling which is not related to the hypofunction of the lymphatic vessel accompanying the fall of expression of VEGFR3 and the inflammation which accompanies this can be provided.

Claims (2)

  A VEGFR3 expression reduction improving agent comprising a red ganoderma extract and / or a black ganoderma extract extracted with carbon dioxide in a temperature range of 20 to 150 ° C and a pressure range of 1 to 80 MPa. A swelling improving agent comprising the VEGFR3 expression lowering improving agent according to claim 1.