JP4037836B2 - Hyaluronidase activity inhibitor - Google Patents

Description

The present invention, high bone (Nuphar japonicum) and / or Nemurokouhone (Nuphar Pumilum) non rhizome part, i.e. the active ingredient an extract of a site other than the rhizomes and be Ruhi Aruronidaze activity inhibitor, and skin external preparation containing the same It is about.

  Conventionally, the rhizome part has been used as a herbal medicinal knives. In addition, as a skin external preparation, the rhizome part powder, water extract, lower alcohol, hydrous lower alcohol extract, etc., for example, as “adhesive plaster” (Japanese Patent Laid-Open No. Sho 62-36328), As a “skin external preparation” for preventing hot flashes after tanning, razor rash, and rough skin (Japanese Patent Application Laid-Open No. 62-51606 (Patent Document 1) below), as a “hair cosmetic” for conditioning hair after washing ( JP-A-63-57313), “cleaning agent” for preventing skin irritation caused by a cleaning agent (JP-A-63-57696), and “cosmetics” expecting an astringent effect (JP-A-64-47708) No. 5) as a “hair conditioner” having an excellent hair nourishing effect (Japanese Patent Laid-Open No. 5-255044), and as a “skin external preparation” used in combination with kojic acid (Japanese Patent Laid-Open No. 7-25762) Permissible literature 2)), as an “interleukin 4 production inhibitor” (JP-A-10-279491 (Patent Document 3 below)) to prevent acne as an external skin preparation or antiallergic agent for atopic dermatitis Function searches are being performed as “acne preventive and therapeutic agents” (Japanese Patent Laid-Open No. 2001-322943) and “hair growth agents” (Japanese Patent Laid-Open No. 2002-47146). Thus, conventionally, only the rhizome part, which is the underground part, has been put to practical use for the kohone and nemuro kone, and the non-rhizome part, which is the above-ground part, has been discarded without being used. For this reason, the functions of non-rhizome extracts of kohone and nemuro kone, particularly solvent extracts other than water, have not been clarified so far.

  By the way, there is a whitening effect as one of the effects expected for external preparations for skin including cosmetics. Conventionally, vitamin C has been used exclusively for imparting a whitening effect to cosmetics, but vitamin C is expected to be most effective as a whitening agent for cosmetics used on the skin due to its light vulnerability. Therefore, as a substitute for that, searching for a tyrosinase inhibitory function in general herbal medicines and selecting and using one having a high inhibition rate is performed. Tyrosinase is an enzyme that promotes the reaction from tyrosine to DOPA and further from DOPA to DOPA quinone in the melanin production system of skin, and DOPA quinone further becomes a melanin pigment through processes such as oxidation, decarboxylation, and polymerization. The suppression is expected to lead to a whitening effect. However, the tyrosinase inhibitory function in actual skin may not be short-circuited to the decrease in melanin production, and a certain whitening agent that suppresses melanin production as an element that impairs whitening has a higher contribution to whitening. That is, the resulting amount of melanin is more likely to help predict whitening function than the trend of tyrosinase attributed to melanin production. For this reason, there are many herbal medicines whose tyrosinase-inhibiting function has been measured, but whitening agents that are actually used in skin applications include arbutin (derived from walrus and cowberry leaves) and kojic acid. There is only.

  On the other hand, regarding the inhibition of hyaluronidase activity, hyaluronidase is a hydrolase of the biomembrane component hyaluronic acid that is present in various parts including lysosomes, testicles, and skin of higher organs, and is active during skin inflammation. To destroy surrounding tissues and facilitate infiltration of inflammatory cells. Therefore, antiallergic action and anti-inflammatory effect can be expected by inhibiting the activity of hyaluronidase. In addition, it is considered that the increased activity of hyaluronidase that decomposes hyaluronic acid, which is a moisturizing component, causes rough skin and roughness, which contributes to wrinkles and looseness. Therefore, by inhibiting the activity of hyaluronidase, it can be expected to prevent moisturizing aging including skin wrinkles and looseness. In the prior art, it has been reported that the water extract of herbal medicine Senkotsu has a hyaluronidase inhibitory action (“inhibitory action of herbal medicines on skin-related enzymes” (Non-patent Document 1 below)), but the technology dealt with here Is also an aqueous extract of a herbal medicine, that is, an aqueous extract of a rhizome of a corn plant, and is not related to an extract of a non-rhizome of a corn plant. In addition, hyaluronidase activity inhibitors are preferably absorbed intradermally in skin preparations such as cosmetics. In terms of intradermal absorption, a non-aqueous solvent extract is more efficient than an aqueous extract. .

Until now, various substances with whitening and hyaluronidase activity have been used as a formulation for cosmetics and other external preparations for skin. The same was true for the extract.
JP-A-62-51606 JP 7-25762 A JP 10-279491 A Yoshiyuki Sawabe and 4 others, “Inhibitory action of crude drugs on skin-related enzymes”, Pharmaceutical Journal, 1998, 118 (9), p. 423-429

An object of the present invention is to provide a suitable heat Aruronidaze activity inhibitor for incorporation into the skin external preparation, also the external preparation for skin which is excellent in heat Aruronidaze inhibitory activity formulated with the hyaluronidase activity inhibitor The purpose is to provide.

The present inventors have had extensive research by focusing on non rhizome of conventionally utilized by high bone and Nemurokouhone that have been discarded without arsenide Aruronidaze activity inhibitory effect surprisingly superior its non-aqueous solvent extract As a result, the present invention has been completed.

That is, the invention according to claim 1 of the present invention is a hyaluronidase activity inhibitor comprising as an active ingredient a non-aqueous solvent extract of non rhizome of high bone and / or Nemurokouhone.

The invention according to claim 2 is a hyaluronidase activity inhibitor comprising, as an active ingredient, an oil-soluble substance extracted from a non-rhizome portion of corn and / or nemur.

The invention according to claim 3 is an external preparation for skin containing the hyaluronidase activity inhibitor according to claim 1 or 2 .

According to the present invention, since it has a heat Aruronidaze inhibitory activity of the extract is excellent from the non rhizome of high bone and / or Nemurokouhone, excellent anti-allergic effect in the skin external preparation, including cosmetics containing this And moisturizing and aging preventive action.

  Further, in the present invention, since the non-rhizome portion of the corn and / or nemuro kone that has been discarded conventionally is used, the waste can be effectively used, and if the non-rhizome portion is used, the rhizome portion is used. In this case, only the non-rhizome portion can be collected, and in this case, the non-rhizome portion can be grown again from the remaining rhizome portion, so that resources can be effectively used.

  Hereinafter, matters related to the implementation of the present invention will be described in detail.

  Nuphar japonicum and / or N. Pumilum is a water lily family, and the root of its root is roasted under the name centrum (river bone) and used for nourishment, improvement of physical condition, irregular physiology, etc. . The actual use of extracts other than rhizomes has not been seen as a crude drug preparation or a drug to be blended in cosmetics and the like.

  In the present invention, a non-rhizome part that is a part other than the rhizome part of such corn and / or nemper, that is, a part corresponding to the above-ground part in a normal plant is extracted with a solvent other than water, and the extract is extracted from the skin. It is used as a medicine to be blended with external preparations. Although the preparation method is not specifically limited, For example, the following extraction methods can be adopted.

  The potato and / or nemuro potato, excluding the rhizome, is washed with water, dried, pulverized and extracted with an extraction solvent by a generally known method. Examples of the drying method include air drying, hot air drying, freeze drying, and the like. Although not particularly limited, freeze drying is preferable.

  The solvent used for extraction is not particularly limited as long as it is a solvent other than water. For example, lower alcohols such as methanol, ethanol, propanol, isopropanol, 1,3-butylene glycol, propylene glycol, dipropylene glycol , Polyhydric alcohols such as glycerin, ethers such as ethyl ether and propyl ether, esters such as ethyl acetate and butyl acetate, ketones such as acetone and ethyl methyl ketone, hydrocarbons such as n-hexane and benzene, chloroform, Examples include halogen solvents such as methylene chloride, and these may be used alone or in combination of two or more. Among these, suitable solvents for obtaining an excellent whitening action include organic solvents having an intermediate polarity such as the ethers, esters, ketones, hydrocarbons and halogen solvents described above, and particularly ethyl acetate and the like. Of these, fatty acid esters such as acetic acid esters are preferred, and in this case, those excellent in both the whitening action and the hyaluronidase activity inhibiting action can be obtained. Thus, when the whitening action and the hyaluronidase activity inhibitory action coexist, the anti-inflammatory action can be expected by the hyaluronidase activity inhibitory action, and thus there is a merit that the whitening effect can be obtained while suppressing rough skin. In view of the fact that many of the conventional whitening agents have irritation, this merit can provide a whitening agent that can be used even for people with weak skin who could not use such whitening agents. Useful in terms. Furthermore, since the effect of preventing wrinkles and sagging of the skin can be expected by inhibiting hyaluronidase activity as described above, the increase of melanin and the increase of wrinkles accompanying aging can be prevented by coexisting this effect with the whitening action. Therefore, it is suitable as an agent for preventing skin aging.

  Examples of the extraction method include dipping or heating under reflux. For example, when extracting by immersion, about 1 to 30 times the amount of solvent may be added to the non-rhizome portion of dried and pulverized corn and / or nemur potato, and immersed at room temperature or under heating. In addition, when heating to reflux, add about 1 to 30 times the amount of solvent to the non-rhizome of dried and pulverized corn and / or Nemuro kone, and extract by refluxing at about 40 to 80 ° C. for about 1 to 10 hours. Can do.

  The extract obtained by extraction with the above solvent can be used by concentrating by an appropriate method, drying and the like. Alternatively, it can also be used as a fraction extract obtained by fractionation using adsorption system chromatography such as silica gel column chromatography. As for the concentration method, distillation or the like can also be used as a suitable method.

  The extract of non-rhizome of corn and / or nemur corn obtained in this way is an oil-soluble substance that is soluble in a non-aqueous solvent and is usually extracted using a non-aqueous solvent as described above. However, in the present invention, the extract is not necessarily limited to a non-aqueous solvent extract, and an oil-soluble substance soluble in a non-aqueous solvent may be extracted using an aqueous solvent. Preferably, the oil-soluble substance is a substance that is soluble in an organic solvent having the above-mentioned intermediate polarity, particularly fatty acid esters including ethyl acetate.

Engaging Ruhi Aruronidaze activity inhibitor of the present invention inhibit is for the active ingredient an extract of the non rhizome of high bone and / or Nemurokouhone obtained as described above, excellent as shown in the following examples Has an inhibitory action on allonidase activity. Incidentally, usage of the human Aruronidaze activity inhibitor is not particularly limited, in paste form, in liquid form, in solid form, also may be in powdered powdered according to conventional methods, are further used for external preparation for skin such as It may be in the form of a solution dissolved in various solvents (for example, polyhydric alcohol).

Further, heat Aruronidaze activity inhibitor is not limited to the case of incorporating the skin external preparation as described below, for example, gum, candy, tablet candy, may be formulated into foods such as beverages, further, oral It can also be used for pharmaceuticals.

  In the external preparation for skin according to the present invention, the amount of the extract of non-rhizome part of the above-mentioned corn and / or nemuro corn is not particularly limited, but is usually 0.0001 to 20% by weight, more preferably 0.8. 01 to 5% by weight.

  In addition to the above essential components, the external preparation for skin includes oils, waxes, hydrocarbons, fatty acids, alcohols, polyhydric alcohols that are usually used according to the type of external preparation for skin, if necessary. Esters, amines / amides / metal soaps, gums / water-soluble polymer compounds, surfactants, antioxidants, vitamins, fragrances, coloring materials, antiseptics, amino acids, UV absorbers, metal ions Blocking agents, anti-inflammatory agents, antihistamines, herbal medicines, other known whitening agents and moisturizing agents, and various other skin chemicals can be blended.

  Examples of the external preparation for skin according to the present invention include, for example, basic cosmetics such as creams, emulsions, lotions, packs, lotions, beauty essences, facial cleansers, makeup cosmetics such as funny and foundation, hair growth And cosmetics such as agents and hair nourishing agents, and may be pharmaceuticals such as ointments, poultices, plasters, and quasi-drugs. The dosage form can be any of the types employed in solutions, emulsions, creams, gels, sols, ointments, sticks, powders, sprays, and other conventional skin external preparations.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by this.

(Examples 1 and 2)
The rhizome part was removed from the whole plant of corn, and the non-rhizome part was air-dried and cut. To 8 kg of this, 60 L of ethyl acetate was added and the mixture was cooled and extracted for 7 days with occasional stirring. This extraction was repeated three times, and the filtered extracts were combined and concentrated to about 1/300, and the concentrated extract was evaporated to dryness to obtain about 350 g of an ethyl acetate extract (sample of Example 1).

  The ethyl acetate extract was fractionated by silica gel column chromatography, and the fractionated liquid was concentrated to obtain about 9.3 g of evaporated to dryness (sample of Example 2).

(Example 3)
In the operation of Example 1 above, ethyl acetate as the extraction solvent was changed to methanol, and the others were extracted and concentrated in the same manner to obtain about 1250 g of an evaporated dry solid (sample of Example 3).

(Test Example 1: Melanin production inhibition test)
Mouse B16 melanoma cultured cells (mouse B16 melanoma 4A5) were used for the melanin production inhibition test. First, 2.5 × 10 ⁵ B16 melanoma cells were seeded in a plastic petri dish having a diameter of 60 mm, and cultured in Eagle's MEM medium containing 10% by weight fetal calf serum at 37 ° C. for 24 hours under 5% CO ₂ . Next, the samples of Examples 1 and 2 were replaced with Eagle's MEM medium containing 10% by weight fetal calf serum added so as to be 20, 10, and 5 μg / mL, respectively, and cultured for 72 hours. After completion of the culture, the cells were detached with trypsin, and the cells were collected by centrifugation. The obtained cells were treated in the order of 5 wt% TCA, ethanol: diethyl ether = 3: 1, diethyl ether, dried, 1N sodium hydroxide containing 10 wt% DMSO was added and dissolved by heating. After cooling, the absorbance at 420 nm was measured, and the amount of melanin was measured. Separately, using the same test with no sample added as a control, the melanin production inhibition rate of the sample was determined by the following formula. In addition to the above, the results are shown in Table 1, comparing the test results of arbutin, which has a reputation for inhibiting melanin production and whitening effect. Although DMSO was used for sample dissolution, it was confirmed that this did not affect the test results.

Inhibition rate (%) = {(A−B) / A} × 100
A: Absorbance when no sample is added B: Absorbance when the sample is added

  As shown in Table 1, the melanin production-suppressing function of the non-aqueous solvent extract from the non-rhizome part of corn was extremely superior to that of arbutin.

(Test Example 2: Hyaluronidase activity inhibition test)
The hyaluronidase activity inhibition test was performed on the samples of Examples 1 and 3 according to a modification of the Morgan-Elson method (Davidson, EA, Aronson, NN: J. Biol. Chem. 242, 437 (1967)).

  Specifically, 0.05 mL of a solution prepared by dissolving bovine testicular-derived hyaluronidase in 0.1 M acetate buffer (pH 4.0) to 2.1 mg / mL and 0.1 mL each of the samples of Examples 1 and 3 were mixed. After incubating at 37 ° C. for 20 minutes, 0.1 mL of a solution prepared by dissolving Compound 48/80, an enzyme activator, in 0.1 M acetate buffer (pH 4.0) to 0.5 mg / mL was added. In addition, it was incubated at 37 ° C. for 20 minutes. Next, 0.25 mL of a solution prepared by dissolving potassium bromide derived from the cock of the chicken, the substrate, in 0.1 M acetic acid buffer (pH 4.0) to adjust to 0.8 mg / mL was added, and incubated at 37 ° C. for 40 minutes. Then, 0.1 mL of 0.4N sodium hydroxide aqueous solution was added to stop the reaction. To this, 0.1 mL of 0.8 M boric acid solution adjusted to pH 9.1 was added and boiled for 3 minutes. After standing to cool, p-DAB solution (10 g of p-dimethylaminobenzaldehyde, 12.5 mL of 10N hydrochloric acid, 87. 5 mL) was diluted 10-fold with acetic acid, 3 mL was added, and the mixture was incubated at 37 ° C. for 20 minutes for color development. Thereafter, the absorbance at 585 nm was measured, and the hyaluronidase activity inhibition rate was determined by the following formula. The results are shown in Table 2. Although DMSO was used for sample dissolution, it was confirmed that this did not affect the test results.

Inhibition rate (%) = {(A−B) − (C−D)} / (A−B) × 100
A: Absorbance at 585 nm of the control solution (after reaction) B: Absorbance at 585 nm of the control solution (before reaction) C: Absorbance at 585 nm of the sample solution (after reaction) D: Absorbance at 585 nm of the sample solution (before reaction)

  As shown in Table 2, it was confirmed that the non-aqueous solvent extract from the non-rhizome part of corn has strong hyaluronidase activity inhibition ability.

  Hereinafter, formulation examples of the external preparation for skin according to the present invention will be shown (all blending amounts are% by weight).

(Formulation Example 1: Cream)
Stearic acid 2.0
Stearyl alcohol 2.0
Reduced lanolin 2.0
Squalane 5.0
Octyldodecanol 6.0
Polyoxyethylene cetyl ether (25EO) 3.0
Lipophilic glyceryl monostearate 2.0
Perfume, preservative, antioxidant Suitable amount Sample of Example 1 0.2
Propylene glycol 5.0
Purified water remaining

(Formulation example 2: lotion)
Citric acid 0.1
Zinc paraphenol sulfonate 0.2
Sorbit 2.0
Glycerin 3.0
Polyoxyethylene oleyl ether (25EO) 1.0
Ethanol 15.0
Perfume and preservatives Appropriate amount Sample of Example 1 0.2
Purified water remaining

(Formulation Example 3: Latex)
Sample of Example 1 0.2
Stearic acid 2.0
Ethanol 1.5
Vaseline 3.0
Lanolin alcohol 2.0
Liquid paraffin 10.0
Polyoxyethylene oleate (10EO) 2.0
Perfume, preservative, antioxidant Suitable amount Glycerin 3.0
Propylene glycol 5.0
Triethanolamine 1.0
Purified water remaining

(Test Example 3: Use effect test)
A test was conducted on the effect of actually using the above-mentioned external preparation for skin. The use test was conducted by 30 healthy adult females 25 to 50 years old as a panelist, and by applying an appropriate amount of the emulsion of Formulation Example 3 to the face for 3 months after washing the face twice daily in the morning and at night. As a control, a formulation prepared by removing the sample of Example 1 from the emulsion of Formulation Example 3 in the same manner was used. The evaluation is performed according to the following criteria, and the results are as shown in Table 3. The numerical values in the table represent the number of people. No one complained of skin abnormalities during the period of use.

・ Effective effect of improving skin wrinkle / sagging: Slightly effective wrinkle / sagging is inconspicuous: Invalid: Wrinkle / sagging slightly inconspicuous: No change from before use.

・ Whitening effect is effective: Dullness of face / stain is slightly inconspicuous: Dullness of face / stain is inconspicuous: Invalid: No change from before use.

  As shown in Table 3, it was confirmed that the skin external preparation according to the present invention significantly reduced skin wrinkles and looseness and had a whitening effect.

(Test Example 4: Use effect test)
Anti-allergic and anti-inflammatory effects were examined using the cream of Formulation Example 1 described above as a skin external preparation. The use test was conducted by applying 30 persons aged 30 to 50 years suffering from eczema, kayumi and rough skin by applying an appropriate amount of the cream of Formulation Example 1 every morning and night for 3 months. As a control, a cream prepared by removing the sample of Example 1 from the cream of Formulation Example 1 by the same method was used. Evaluation is performed according to the following criteria, and the results are as shown in Table 4. The numerical values in the table represent the number of people. No one complained of skin abnormalities during the period of use.

・ Eczema, rough skin improvement effect Effective: Eczema / Kayumi / Skin rough improved slightly effective: Eczema / Kayumi / Skin rough improved slightly: Invalid before use and no change.

  As shown in Table 4, it was confirmed that the external preparation for skin according to the present invention significantly improved eczema, kayumi and rough skin.

Engaging Ruhi Aruronidaze active inhibitor in the present invention, including the external preparation for skin, such as foods and oral drugs, a variety of anti-allergic effect or anti-inflammatory effect by heat Aruronidaze inhibitory activity, wrinkles, sagging preventive effect sought Available for use. Moreover, the skin external preparation which concerns on this invention can be utilized as cosmetics, a pharmaceutical, a quasi-drug, etc.

Claims (3)

  A hyaluronidase activity inhibitor comprising, as an active ingredient, a non-aqueous solvent extract of non-rhizome part of corn and / or nemuro corn.   A hyaluronidase activity inhibitor comprising as an active ingredient an oil-soluble substance extracted from a non-rhizome part of corn and / or nemur. An external preparation for skin, comprising the hyaluronidase activity inhibitor according to claim 1 or 2 .