TW201340987A - Promoter for expression of hyaluronic acid synthase gene - Google Patents

Abstract

To provide a substance that promotes the expression of a hyaluronic acid synthase gene in an effective manner and is able to be used in a wide range of applications. The promoter for the expression of a hyaluronic acid synthase gene has at least one active ingredient selected from the group consisting of: beta-caryophyllene, caryophellene oxide, and a compound having the following structural formula. (X is CH3, NH2, or NHCH3.)

Description

Performance enhancer for hyaluronic acid synthase gene

The present invention relates to a performance enhancer for a hyaluronic acid synthase gene.

Hyaluronic acid is a high-molecular polysaccharide which is present in the skin (epidermal and dermal), cartilage, joint fluid, etc., and has a retention of water in the intercellular space, and maintenance of cells based on the formation of a gel-like matrix in the tissue. Various functions such as the maintenance of lubricity and softness of the tissue, resistance to external forces such as mechanical damage, and prevention of bacterial infection. Hyaluronic acid is abundant in the skin, and its ability to retain water is very high. It is closely related to the moisturizing, firming and elasticity of the skin. It is believed that the amount of hyaluronic acid decreases with age, and the firming of the skin is attenuated, and wrinkles or relaxation occur. Wait for skin aging. Therefore, in order to maintain moisturizing or firming skin, hyaluronic acid is added to cosmetics and the like, and hyaluronic acid is added from the outside. In recent years, the development of a drug that promotes hyaluronic acid synthesis in the skin has been attracting attention not only by supplying hyaluronic acid to the surface of the skin.

Further, it is known that the healing process after burn injury, the hyaluronic acid in the granulation is significantly increased during the period from the initial stage of the proliferating granulation tissue under the free necrotic tissue to the entire tissue being replaced by the granulation tissue (Non-Patent Document 1), and the hyaluronic acid production promoter is also It is expected as a therapeutic drug in the early stage of burns.

For example, it is reported that Cactus fruit juice obtained from the fruit of the plant belonging to the genus Opuntia, plant body of Phyllanthus emblica or its extract, Curcuma amada ROXB. The plant body or the extract thereof is equivalent to the promotion of hyaluronic acid production in human dermal fibroblasts (Patent Documents 1 and 2).

Regarding hyaluronic acid, there are reported enzymes which are involved in the synthesis of hyaluronic acid in the skin, which are contained in the stratum corneum, the epidermis and the dermis, and there are three kinds of hyaluronic acid synthase (HAS (Hyaluronan Synthase) 1, HAS2, HAS3). Further, it is known that the hyaluronic acid produced by the hyaluronic acid synthetase has a molecular weight of about 10 ³ Da to 10 ⁷ Da, and exhibits various biological or physical properties due to the difference in molecular weight. It is believed that the difference in molecular weight of hyaluronic acid affects physical properties such as water retention and viscoelasticity, especially the high-moisture hyaluronic acid hyaluronic acid is closely related to the skin aging phenomenon such as skin firming, wrinkle or relaxation. Further, it is also known that the polymer hyaluronic acid in the dermis or the epidermis is mainly produced by hyaluronic acid synthetase 2 (HAS2), for example, it is known that (-)-mucilone promotes the expression of the Has2 gene (for example, refer to Non-Patent Document 2, Non-Patent Literature). 3).

Furthermore, an extract of a plant belonging to the genus Equisetum was reported to increase the expression of HAS2 mRNA (Messenger Ribonucleic Acid) in normal human epidermal keratinocytes, and promote the production of high molecular hyaluronic acid in epidermal cells (Patent Document 3) ).

Furthermore, it is reported that essential oils such as juniper oil, eucalyptus oil, and cedar oil are used in normal human skin fibroblasts to guide the circadian rhythm of the hyaluronic acid synthase gene, and in the evaluation system, the Has2 gene is promoted. Performance (Patent Document 4).

As described above, by promoting the hyaluronic acid synthase gene, particularly the hyaluronic acid synthetase 2 gene (Has2) which contributes to the production of hyaluronic acid, skin aging prevention (maintaining skin firmness or elasticity), Moisture, prevention and improvement of arthritis, treatment in the early stage of scald, etc., and prevention or improvement of various symptoms and diseases which are beneficial to the promotion of hyaluronic acid. Therefore, the industry is further strongly expected to develop novel agents that can be used in a wide range of applications and that can effectively promote the performance of hyaluronic acid synthetase genes.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open Publication No. 2011-195473

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2010-229111

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2011-195493

[Patent Document 4] International Publication No. 2011/122040

[Non-patent literature]

[Non-Patent Document 1] "Combining Organizations and Diseases", Kodansha, 153 pages, 1980

[Non-Patent Document 2] Sakai S. et al., Skin Pharmacol. Appl. Skin Physiol., 12, 276-283 (1999)

[Non-Patent Document 3] Sayo T. et al., Skin Pharmacol Physiol, 17, 77-83 (2004)

The present invention has been made in view of the above circumstances to provide a novel pharmaceutical agent which can promote the expression of a hyaluronic acid synthase gene.

The present inventors succeeded in culturing human skin fibroblasts to guide the circadian rhythm of the expression of the hyaluronic acid synthetase gene, and thereby, by using the system to evaluate the gene expression at a higher time point, the test substance can be evaluated with high sensitivity. Hyaluronic acid synthetase gene expression promoting effect. The present inventors have found that β-caryophyllene, caryophyllene oxide, and methyl benzoate derivative can promote the expression of the hyaluronic acid synthetase gene in the evaluation system, thereby completing the present invention.

The hyaluronic acid synthase gene expression promoter of the present invention contains, as an active ingredient, one or more selected from the group consisting of β-caryophyllene, caryophyllene oxide, and a compound having the following structural formula: [Chemical Formula 1]

X is CH ₃ , NH ₂ or NHCH ₃ .

To date, these specific compounds have been shown to promote the performance of the hyaluronic acid synthase gene.

As a human hyaluronic acid synthase, there are HAS1, HAS2, and HAS3, but the hyaluronic acid having high moisturizing power is mainly produced by HAS2. Therefore, the hyaluronic acid synthase gene expression promoter of the present invention is preferably a gene for promoting HAS2 (Has2). Performer.

The hyaluronic acid synthase gene expression promoter of the present invention can be used in various forms such as medicine, quasi-drugs, cosmetics, foods, groceries, clothing, etc., and can safely and effectively improve the performance of the hyaluronic acid synthase gene, and prevent skin aging ( To prevent or improve the skin, the moisturizing, the moisturizing sensation, the prevention and improvement of arthritis, etc., and the initial treatment of scald, etc., to treat or improve various symptoms or diseases that benefit from the promotion of hyaluronic acid. Wait.

Figure 1 is a graph showing the circadian rhythm of the expression of the hyaluronic acid-synthesizing gene using cortisol in cultured human skin fibroblasts.

Fig. 2 is a graph showing the expression of a hyaluronic acid synthetase gene by a test substance in cultured human skin fibroblasts.

The hyaluronic acid synthase gene expression promoter of the present invention contains β-caryophyllene, caryophyllene oxide or the above methyl benzoate derivative as an active ingredient. These compounds are all known compounds and will be briefly described below.

Β-caryophyllene (4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undecene) is a sesquiterpenoid having a molecular weight of 204.36 represented by the following structural formula, and a commercial product. Mostly separated by clove oil. It is known to be naturally present in clove oil, bitter baba oil, lavender oil, and the like.

Caryophyllene oxide ((1R,4R,6R,10S)-4,12,12-trimethyl-9-methylene-5-oxatricyclo[8.2.0.04,6]dodecane) The sesquiterpenoids having a molecular weight of 220.35 represented by the following structural formula can be synthesized by monoepoxidation of β-caryophyllene with a peroxyacid. It is known to be naturally present in clove oil, lavender oil, and the like.

Further, methyl 2-methylbenzoate which can be used as the methyl benzoate derivative in the present invention is a colorless liquid having a molecular weight of 150.18. Methyl 2-aminobenzoate is a liquid having a molecular weight of 151.17, which can be obtained by adding 80 parts of o-aminobenzoic acid to 120 parts of methanol, and slowly adding 100 parts of concentrated sulfuric acid to 40 degrees or less, and 75 ~83 degrees After completion of the reaction and recovery of methanol, the mixture was added to a solution of 110 parts of sodium carbonate and 240 parts of water for neutralization and washing with water, followed by purification by distillation. There are also synthetic methods obtained from o-nitrotoluene. It is known to be naturally present in neroli oil, perfume tree oil, and the like. Methyl 2-methylaminobenzoate is known as a colorless liquid having a molecular weight of 165.2, and is naturally present in orange peel, citrus oil, and the like.

The hyaluronic acid synthase gene expression promoter of the present invention comprises one or more of the above compounds. In the present invention, the above compound may be derived from a natural one or may be a synthetic.

The hyaluronic acid synthase gene expression promoter of the present invention can be used alone or in combination with other agents having a hyaluronic acid synthase gene expression promoting effect.

Further, the hyaluronic acid synthase gene expression promoter of the present invention may be used alone or in various subjects. The type of the object may further contain any constituent elements in addition to the above-described essential components.

For example, when the object is a skin external preparation, it may be according to its dosage form (for example, a liquid, a powder, a granule, an aerosol, a solid, a gel, a patch, a suppository, etc.), or a product. The form (for example, a cosmetic, a pharmaceutical, a quasi-drug, etc.) contains any of the components contained in the usual external preparation for skin and the hyaluronic acid synthase gene expression promoter. Skin external preparations include all concepts applicable to the skin (including scalp, hair, nails), including, for example, basic cosmetics, makeup cosmetics, hair cosmetics, skin or hair cleansing. Cosmetics such as materials, or various pharmaceuticals or quasi-drugs such as ointments, patches, suppositories, and toothpastes. The dosage form is not particularly limited, and includes, for example, an aqueous solution, a solubilized system, an emulsified system, an oil system, a gel system, a cream system, an ointment system, an aerosol system, a water-oil two-layer system, and a water-oil- Any dosage form such as three layers of powder. When the external preparation for the skin is a cosmetic, examples thereof include perfumes, eau de toilette, cologne, creams, lotions, lotions, foundations, powders, lipsticks, soaps, shampoos, and shampoos. Shower gel, body lotion, body powder, body wash, etc.

Moreover, it can be used in any kind of groceries such as a fragrance, a deodorant, a scented candle, a scented incense, a stationery, a wallet, a bag, a shoe, or any clothing such as underwear, a suit, a hat, a stocking, a sock, and the like. Alternatively, the hyaluronic acid synthase gene expression promoter of the present invention may be contained in various forms of supplements such as powders, granules, troches, capsules, and the like (nutrition-assisted foods), and any foods such as cakes and drinks, and may also be used for inhalation. Inhaled products such as pharmaceuticals or space dispersing agents.

In addition, although the use form of the hyaluronic acid synthase gene expression promoter of the present invention is exemplified, it is not limited thereto, and it can be used in any form as long as it can attain the effect of the invention. Further, as long as the effects of the present invention are not impaired, in addition to the hyaluronic acid synthase gene expression promoter of the present invention, other agents having a hyaluronic acid production-promoting action can be formulated according to specific conditions.

The amount of the hyaluronic acid synthase gene expression promoter of the present invention in the object can be appropriately selected depending on the type, form, and object of the compound to be used, and is not particularly limited. For example, the total mass of the object is 0.00001. ~100% by mass, more preferably 0.0001 to 50% by mass, still more preferably 0.0001 to 20% by mass.

Further, the specific application of the hyaluronic acid synthase gene expression promoter or the object to be formulated according to the present invention is not particularly limited, and for example, it can be used for preventing aging of the skin (maintaining firmness or elasticity of the skin), moisturizing, and arthritis. Prevention, improvement, etc., treatment of the initial stage of scald, etc., prevention and improvement of various symptoms and diseases that benefit from the promotion of hyaluronic acid.

[Examples]

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited by the following examples. Further, as the hyaluronic acid synthase gene of human beings, Has1, Has2, and Has3 are known, but the expression of the human hyaluronic acid synthetase 2 gene (hHas2) is representative in this example. The test substances used in the following are used by purchasing a commercially available compound.

Guide to the circadian rhythm of hyaluronic acid synthase gene expression in human skin fibroblasts

It was confirmed that the circadian rhythm of the expression of the hyaluronic acid synthetase gene (hHas2) can be guided in a system using cultured human skin fibroblasts.

The human skin fibroblast cell line was purchased and purchased from normal adult skin fibroblasts (Cell Application, Inc) and used for experiments. Sowing it to 10% FBS (Foetal Bovine Serum, fetal bovine serum), 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 4-(2-hydroxyethyl)-1-piper The medium of DMEM (Dulbecco's Modified Eagle's Medium) of ethanesulfonic acid), Glutamax, and an antibacterial agent was cultured at 37 ° C under 5% CO ₂ . On the sixth day of culture, after adding cortisol 50 ng/ml for 2 hours, the cells were sampled over time. RNA was extracted from cells using a commercially available RNA extraction kit, using a commercially available PCR (Polymerase Chain Reaction) primer (Perfect Real Time Primer, TAKARA BIO), by RT-PCR (Reverse Transcription- The amount of hHas2 gene expression was determined by Polymerase Chain Reaction, reverse transcription-polymerase chain reaction. In addition, the amount of expression of RPLP0 (Ribosomal Protein Large P0), which is a housekeeping gene, was quantified and used as an internal standard, and the relative expression amount of the target gene with respect to the expression amount of RPLP0 was calculated.

It is considered that in the living body, glucocorticoids such as cortisol are involved in the regulation of the circadian clock such as peripheral tissues, and when the morning is waking up, the concentration of blood in the cortisol rises and the circadian clock is reset. In cultured cells, usually each cell line runs rhythms at different timings, but by stimulating with a signal stimulating factor such as cortisol, it is also possible to guide circadian rhythms in cultured cells.

The results of measuring the performance of hHas2 are shown in Fig. 1. It was confirmed that by stimulating with cortisol, the circadian rhythm of about 24 hours was performed to express hHas2.

Evaluation of the effect of hyaluronic acid synthase gene expression

Based on the above results, in the cultured human skin fibroblast evaluation system, it was confirmed that hHas2 operates the circadian rhythm using the stimulation produced by cortisol. Since it is preferable to evaluate in the time period in which the amount of expression is high, the gene expression amount of the hyaluronic acid synthase gene of the test substance is evaluated by using the gene expression amount after 16 hours of stimulation as an index.

The fibroblasts derived from normal adult skin (Cell Application, Inc) were seeded by the same method as described above, and the respective agents were added at a dose of 50 ppm on the sixth day of culture, and the cells were subjected to 16 hours later. sampling. Further, as a control, an equal amount of ethanol was added instead of the test substance. RNA was extracted from the cells using a commercially available RNA extraction kit, and the amount of expression of the hHas2 gene was determined by RT-PCR using a commercially available PCR primer (Perfect Real Time Primer, TAKARA BIO). In addition, the amount of expression of RPLP0 as a housekeeping gene was quantified and used as an internal standard, and the relative expression amount of the target gene with respect to the expression amount of RPLP0 was calculated. A multi-comparison test of Dunnett was performed on the obtained value, and it was judged that it was effective to have a significant difference of 5% risk on one side compared with the control.

The relative expression amount of the hHas2 gene after 16 hours of addition of the test substance is shown in Fig. 2 .

Further, the relative expression amount of the hHas2 gene after 16 hours is shown in Table 1 below.

Shows β-caryophyllene, caryophyllene oxide, methyl 2-methylbenzoate, methyl 2-methylaminobenzoate, and methyl anthranilate (methyl anthranilate) Compared with the control, the hHas2 gene expression was significantly increased, and the compounds promoted the performance of the hyaluronic acid synthase gene.

(mixing example)

Hereinafter, the preparation example of the hyaluronic acid synthase gene expression promoter of the present invention is shown, but the practice of the present invention is not limited to the following. Further, in the following, the blending amount means the mass % relative to the total amount of the product.

Fragrance

Indoor fragrance

Aromatherapy

Bathing agent

Massage gel

Massage cream

Aromatic fiber

In the copper ammonium cellulose solution (cellulose concentration: 10% by weight, ammonium concentration: 7% by weight, copper concentration: 3.6% by weight), the inclusion of the present invention in the range of 0.1 to 20% by weight based on the weight of the cellulose is added. The microcapsules of the gene expression promoter (having a particle diameter of 50 μm or less and the ratio of the compound to the microcapsules of 50% by weight) are mixed and then spun according to a usual wet spinning method, and are subjected to a scouring step and a drying step. Aromatic fibers are obtained.

Granule

Lozenge (chewing type)

tablet

candy

chewing gum

The preparations of the preparation examples can promote the performance of the hyaluronic acid synthase gene and increase the production of hyaluronic acid according to the use test in the typical use form of each product form.

Claims (4)

A hyaluronic acid synthase gene expression promoter comprising, as an active ingredient, one or more selected from the group consisting of β-caryophyllene, caryophyllene oxide, and a compound having the following structural formula: X is CH ₃ , NH ₂ or NHCH ₃ . The hyaluronic acid synthase gene expression promoter according to claim 1, wherein the hyaluronic acid synthase gene is Has2. The use of a compound for producing a hyaluronic acid synthase gene expression promoter, wherein the compound is selected from the group consisting of β-caryophyllene, caryophyllene oxide, and a compound having the following structural formula: X is CH ₃ , NH ₂ or NHCH ₃ . The use of claim 3, wherein the hyaluronic acid synthetase gene is Has2.