RU2420264C2 - Preparation for local application on skin, which contains flavanon derivative - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to creation of preparations for local application on skin with wound-healing effect. In order to increase ability of live body to tissue regeneration, such as ability of fibroblast to collagen production in derma in injured skin region, for instance, such as large wrinkle or wound, as active component of preparation for local application on skin, flavanon derivative, such as farrerol, can be applied. Additionally, for efficient detection of substance, which has excellent effect on wound healing acceleration, reconstructive action of collagen fibre bundles was tested with application of skin wound model.

EFFECT: creation of preparation with wound-healing effect.

8 cl, 11 ex, 7 tbl

Description

FIELD OF TECHNOLOGY

The present invention relates to a preparation for topical application to the skin, and more particularly, to a preparation for topical application to the skin containing a flavanone derivative. The present invention also relates to a method for enhancing the ability of fibroblast to produce collagen. Further, the present invention relates to a method for screening a substance having a wound healing effect.

BACKGROUND

The skin consists of a keratinized layer, epidermis, basement membrane and dermis. It is believed that the structure of the dermis has a significant effect on the properties of the "skin" that can usually be detected. It is also believed that one of the important components of the dermis are collagens, which have a significant effect on the properties of elasticity and other things. Derma collagens include collagen I, collagen III, collagen IV, collagen V, collagen VII, and the like, and have their own important functions. These collagens are formed from the collagen produced by fibroblasts (see, for example, Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 3). A decrease in the ability to produce collagen (including procollagen) by fibroblasts is also known to occur due to aging and damage caused by UV rays, and is responsible for impaired skin function. In other words, an important factor in restoring skin function is restoring the ability of fibroblast to produce collagen after damage.

The appearance of a person changes with age and exhibits signs of aging. One of such signs is the formation of wrinkles on the skin, and the main objective of cosmetic preparations is to prevent the formation of such signs. It is believed that one of the evolutionary mechanisms of wrinkle formation may include the destruction of the structure of bundles of collagen fibers of the dermis or the violation of such a structure. Sometimes, the destruction or violation of the structure of the bundles of collagen fibers of the dermis leads to a partial loss of the bundles of collagen fibers of the dermis themselves. Part of the skin in this condition may have a structure similar to that of wounded skin and is usually denoted by the term "large wrinkle". As is known, terpenes, such as monoterpene or triterpene, or a derivative thereof, are used to restore bundles of collagenic fibers of the dermis to correct the destruction or structural disturbance of bundles of collagenic fibers of the dermis (see, for example, Patent 1). In addition, it is also known that some of the plant extracts have a corrective effect on bundles of collagenic fibers of the dermis (see, for example, Patent 2 and Patent 3). Among plant extracts, Hypericum extract is known to be excellent in terms of the corrective action on bundles of collagenic fibers of the dermis (see, for example, Patent 4). These terpenes and extracts are effective in enhancing the ability of fibroblast to produce collagen and, if bundles of collagen fibers are localized around, they can extend bundles of collagen fibers along such a bundle of fibers. However, these substances cannot create bundles of collagen fibers satisfactorily when the bundles of collagen fibers of the dermis themselves are not present in sufficient quantity and are absent around fibroblasts. Therefore, the aforementioned terpenes and extracts cannot satisfactorily accelerate the correction of bundles of collagen fibers in a segment in which bundles of collagen fibers, such as large wrinkles, are absent. Therefore, there is a problem that a sufficient improvement effect cannot be achieved with respect to the aforementioned symptom.

In addition, the skin is both an important tissue for protecting the living body and the respiratory organ, so any skin defect will be dangerous both for the protective mechanism and the respiratory function of the living body. Thus, there is a proven theory that the loss of one third of the epidermis due to injuring burns or the like leads to a danger to life. In this context, for many years there has been a demand for the development of technology for the rapid regeneration of damaged skin segments. Wound healing agents that are known in the art include, for example, Curcuma extract and lactoferrin (see, for example, Patent 5 and Patent 6). However, any of these substances has an insufficient effect of regeneration of skin tissue, since there is no corrective effect on the structure of bundles of collagenic fibers of the dermis. In such circumstances, inulin (see, for example, patent 7), collagen hydrolysates (see, for example, patent 8), lactoferins (see.) Were investigated as substances for wound healing to activate cell growth and enhance their collagen productivity. , for example, patent 6) and so on. The wound healing effect of these substances was confirmed through activity in the regeneration of skin tissue or the expression of RNA enzymes, or the like, included in the proliferative activity of fibroblasts. However, any phenomenon that clearly leads to the actual regeneration of the skin through the action of these substances was not observed at all. Further, with respect to these substances, no effect was observed at all on the reconstruction of the damaged part of the living body, for example, with the lack of the structure of the bundles of collagen fibers caused by the wound.

Thus, no substance was found that has a sufficient effect on tissue regeneration, wrinkle improvement, wound healing, and so on, in living body tissue with a part damaged by the loss of collagen fiber bundle structure or similar disorders.

On the other hand, farrerol is a compound having a flavonone structure and is known to be found in various plants, and a method for its synthesis is already known (see, for example, patent 9, patent 10, and patent 11). However, the technology for incorporating it into a preparation for topical application to the skin was completely unknown. In addition, any corrective effect on the bundles of collagenic fibers of the dermis and the effect of accelerating wound healing were completely unknown.

Further, a study of co-culture showed that the action of keratinocytes can cause an improvement in the ability of fibroblast to produce collagen (see, for example, non-patent document 4). However, any substance affecting keratinocytes in the direction of enhancing the ability of fibroblast to produce collagen was not known.

On the other hand, with regard to the ability of fibroblast to produce collagen, any of the aforementioned wound healing substances, such as inulin, collagen hydrolysates and lactoferrins, have been investigated as a substance to enhance the ability of fibroblast to produce collagen. However, an improvement in the ability of fibroblast to produce collagen is a necessary but not sufficient condition to accelerate wound healing. As of today, there is a substance that is ineffective in accelerating wound healing, even having the ability to improve collagen production.

In addition, a method for screening the effectiveness of accelerating wound healing also involves testing the migration activity of fibroblasts (see, for example, Non-Patent Document 5). In this method, a collagen gel is prepared using fibroblasts and then a defective segment is created in it, followed by measuring the migration of fibroblasts into the defective segment. However, improving the migratory ability of fibroblasts is a necessary, but not sufficient, condition for accelerating wound healing. Therefore, there is a component without any effect on the acceleration of wound healing, even if it is examined in relation to the migration ability of fibroblasts.

In other words, these screening methods are not suitable for detecting a substance having both the effect of improving the ability of fibroblast to produce collagen and the effect of improving the migratory ability of fibroblasts, which is required to accelerate wound healing. In such circumstances, there is a need to develop a technology for producing a substance having an excellent wound healing acceleration effect.

[Patent 1] JP 2002-104921A

[Patent 2] JP 2002-29988A

[Patent 3] JP 2002-29987A

[Patent 4] JP 2002-29986A

[Patent 5] JP 11-199461A

[Patent 6] JP 07-196529A

[Patent 7] JP 2004-501176A

[Patent 8] JP 2003-137807A

[Patent 9] WO 02/092110

[Patent 10] EP 122053A

[Patent 11] JP 58-21678A

[Non-Patent Document 1] Keene et al., J. Cell. Biol., 104, 611-621 (1987)

[Non-Patent Document 2] Shimizu et al., Lab. Invest., Jun., 76 (6), 753-63 (1997)

[Non-patent document 3] Vazquez F. et al, Maturitas, 25, 209-15 (1996)

[Non-Patent Document 4] Eming S.A. et al., Hum. Gene Ther., 9 (4), 529-39 (1998)

[Non-Patent Document 5] Roman O'Leary, Mark Rerek, and Edward John Wood, Biol. Pharm. Bull., 27 (2), 266-270 (2004)

SUMMARY OF THE INVENTION

The present invention relates to a preparation for topical application to the skin, having an excellent corrective effect on the damaged structure of bundles of collagen fibers of the dermis (hereinafter, also simply referred to as "bundles of collagen fibers"). In particular, the present invention relates to a technology for accelerating wound healing in a damaged area of the skin, for example, in a large wrinkle or wound, by enhancing the ability of a living body to regenerate tissue, which is represented, for example, by the ability of fibroblast to produce collagen, or the like, in the dermis.

Further, the present invention relates to a technology for regenerating skin tissue. In particular, the present invention relates to a technology for enhancing the ability of fibroblast to produce collagen by acting on keratinocytes.

Further, the present invention relates to a method for screening a substance having an excellent wound healing acceleration effect.

The inventors of the present invention searched for a substance that could be included in a topical skin preparation having effects of enhancing the ability of fibroblast to produce collagen and accelerating wound healing, and ultimately found that flavanone derivatives such as farrerol could have an excellent effect on in this regard. In addition, the inventors of the present invention also found that flavanone derivatives can affect keratinocytes with respect to enhancing the ability of fibroblast to produce collagen. Further, the inventors of the present invention completed the present invention by establishing that cosmetics containing flavanone derivatives have excellent wrinkle improving effects and topical preparations containing flavanone derivatives have an excellent effect on accelerating wound healing.

In addition, the authors of the present invention completed the present invention by establishing that a substance having the effect of accelerating wound healing can be screened efficiently by testing the corrective effect on collagen fiber bundles in a method using a skin wound model in the search for a substance having accelerated activity wound healing.

Thus, the present invention is described below.

[1] First, the invention relates to a preparation for topical application to the skin (hereinafter also referred to as "a preparation for topical application to the skin of the present invention") containing a flavanone derivative represented by the following general formula (1), and / or its salt.

General formula (1)

In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydrogen atom or an alkyl group having 1-4 carbon atoms.

The flavanone derivative is preferably farrerol.

Farrerol

Further, in a preferred embodiment, the topical skin preparation of the present invention comprises an extract of Hypericum erectum, Guttiferae, containing from 10 ^-6 to 0.1% by weight of the flavanone derivative and / or its salt.

In addition, a topical skin preparation of the present invention can be used to normalize the structure of the skin. In particular, it is preferably used to correct damaged bundles of collagenous fibers of the dermis. Further, the preparation is preferably used to facilitate the regeneration of the damaged area of the skin and the closure of the damaged area with regenerated tissue and the healing of the wound. In addition, it is also preferable to use it to enhance keratinocyte activity to increase the ability of fibroblast to produce collagen.

[2] Secondly, the invention relates to the use of a flavanone derivative represented by the general formula (1) and / or its salt in the preparation of a preparation for topical application to the skin.

[3] Thirdly, the invention relates to a method for normalizing the structure of the skin, comprising introducing into the skin a preparation for topical application to the skin, which contains a flavanone derivative represented by the general formula (1) and / or its salt.

In the aforementioned second and third aspects of the invention, preferred embodiments of the flavanone derivative and preferred use of the preparation for topical application to the skin are the same as in the first aspect of the invention.

[4] Fourth, the invention relates to a method for enhancing the action of keratinocytes to increase the ability of fibroblast to produce collagen, where the method includes the introduction of a derivative of flavanone represented by the general formula (1) and / or its salt, under the conditions of co-existence of red blood cells with fibroblasts.

A preferred embodiment of the flavanone derivative is the same as in the first aspect of the invention.

Administration may be transdermal administration. More specifically, the flavanone derivative and / or its salt is preferably contained in a preparation for topical application to the skin and then applied to the skin.

[5] Fifth, the invention relates to a method for screening a substance having a wound healing effect, where the method includes:

culturing normal animal fibroblasts in the presence of a test substance;

preparation of collagen gel by adding a solution of collagen medium to the resulting culture;

culturing fibroblasts in a collagen gel in the presence of a test substance;

the formation of a damaged area in a collagen gel;

the cultivation of fibroblasts in a co-existence of a collagen gel having a damaged area with the test substance;

and observing the extent to which the damaged area is filled or restored during cultivation (hereinafter also referred to as the “screening method of the present invention”).

Further, in the screening method of the present invention, a test substance having the effect of accelerating wound healing can be tested by comparing the degree of filling or restoration of the damaged area in the presence of the test substance and the degree of filling or restoration of the damaged area in the absence of the test substance.

In particular, when the degree of filling or restoration of the damaged area in the presence of the test substance is greater than the degree of filling or restoration of the damaged area in the absence of the test substance, it can be considered that the test substance has the effect of accelerating wound healing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart illustrating the results obtained in Example 1, in which chart 1 represents the relationship between the number of days of incubation and the number of cells in the absence of heparin treatment, chart 2 represents the relationship between the number of days of incubation and the number of cells in the presence of a treatment of 0.01% - heparin.

FIG. 2 is a group of photographs of the behavior of fibroblasts observed in the wound healing model according to Example 2.

FIG. 3 is a group of photographs characterizing the construction of bundles of collagen fibers observed in Example 3.

FIG. 4 is a group of photographs of the status of the damaged area of the collagen gel observed in Example 6.

FIG. 5 is a diagram illustrating the results obtained in experiment 1 of Example 9, in which absorbance values (450 nm) are presented as the response value to the addition of solutions of farrerol of appropriate concentrations.

FIG. 6 is a diagram illustrating the results obtained in experiment 2 of Example 9, in which keratinocyte growth rates are presented as the magnitude of the response to the addition of solutions of farrerol of appropriate concentrations.

FIG. 7 is a series of photographs of the keratinocyte species observed in experiment 2 of Example 9.

FIG. 8 is a diagram illustrating the results obtained in experiment 3 of Example 9, in which the amount of procollagen obtained is shown as the response to the addition of solutions of farrerol of appropriate concentrations.

FIG. 9 is a series of photographs of the condition of a damaged area of a collagen gel observed in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

(A) A flavanone derivative and a salt thereof used in the present invention

The flavanone derivative that can be used according to the present invention is any of the compounds represented by the general formula (1).

In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydrogen atom or an alkyl group having 1-4 carbon atoms.

At least one of R1, R2, and R4 is preferably a hydrogen atom, and, in particular, all of the three substituents are preferably hydrogen atoms. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group and a butyl group. Of these, in particular, a methyl group is preferred.

Further, at least one of R3 and R5 is preferably an alkyl group having 1-4 carbon atoms, and in particular, both of the substituents are preferably alkyl groups having 1-4 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group and a butyl group. Of these, a methyl group is more preferred. In particular, both R3 and R5 are preferably methyl groups.

Examples of such flavanone derivatives include farrerol, methylfarrerol and dimethylfarrerol.

Flavanone derivatives can be synthesized from known compounds according to JP 58-21678A. In addition, they are also present in plants, so they can also be isolated and purified from plant extracts. Farrerol, one of the compounds represented by the general formula (1), can be isolated and purified from the extract of the Hypericum erectum plant, Guttiferae. An example of a method for its preparation will be described below. It is not necessary to say that when a plant-derived flavanone derivative, and / or its salt, is contained in a topical preparation for skin application, as described below, an extract containing a predetermined concentration of the flavanone derivative and / or its salt, sufficient to achieve the desired effect.

Production Example 1

10 l of an 80% aqueous ethanol solution was added to 1 kg of the dried aerial part of Hypericum erectum Thunberg (Guttiferae) and then refluxed for 4 hours with stirring. After cooling, the insoluble residue was removed by filtration and then concentrated under reduced pressure, followed by freeze drying. The result was 51 g of amorphous substance. This product was dispersed in water, placed in a DIAION HP20, washed with a stream of 5 L of water and 5 L of a 50% aqueous solution of ethanol, in that order, and washed with 99% ethanol. The resulting eluate was fractionated. Then, the fractions obtained in this way were further subjected to thin-layer chromatography and HPLC to control their content, in order to jointly combine fractions containing the same individual substance. On the other hand, fractions comprising two or more components were further purified by re-chromatography. The obtained fraction containing one component was investigated for its effect with respect to the reconstruction of bundles of collagen fibers. Then, the structure of an individual component from the fraction exhibiting such a strong effect was identified using proton NMR, elemental analysis, mass analysis, and ¹³ C-NMR. As a result, the component was farrerol with a yield of 3.2 mg. In addition, about 0.006% by weight of farrerol was included in the product recovered by the solvent, which was obtained from material extracted with 80% aqueous ethanol.

Preferred examples of the salt of the flavanone derivative include, without limitation, if they are physiologically acceptable, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; organic amine salts such as ammonium salt, triethylamine salt, triethanolamine salt and monoethanolamine salt; salts of basic amino acids such as lysine salt and arginate.

The aforementioned flavanone derivative and its salt have the effect of accelerating the ability of fibroblasts to reconstruct the structure of bundles of collagenic fibers of the dermis. More specifically, the flavanone derivative allows fibroblasts to migrate to any area at the same level where collagen fiber is not present and at the same time increases the ability of fibroblast to produce collagen per se, thereby correcting bundles of collagen fibers in the damaged tissue area. Further, wound healing can be accelerated by accelerating the regeneration of skin tissue in the damaged area of the skin and enhancing the ability of the regenerating tissue to cover the damaged area.

(B) A topical skin preparation of the present invention

A characteristic feature of a topical skin preparation of the present invention is the presence of a flavanone derivative and / or its salt. For example, the preparation may contain a flavanone derivative and / or its salt in the form of a plant extract. The flavanone derivative may preferably be farrerol. Preferably, a topical skin preparation of the present invention may include a Hypericum erectum extract containing farrerol.

As described above, the flavanone derivative and its salt have the effect of correcting the structure of bundles of collagenic fibers of the dermis. Thus, a topical skin preparation of the present invention can be used to normalize the structure of the skin. The phrase "normalization of the structure of the skin" here refers to a lesser degree of violation of the structures of the epidermis, dermis and subcutaneous fat that form the skin. More specifically, for example, it includes the correction of the damaged structure of bundles of collagenous fibers of the dermis. The phrase “correction of the damaged structure of the bundles of collagenic fibers of the dermis” here refers to the construction of the structure of bundles of collagenic fibers of the dermis in the region where the structure of the bundles of collagenic fibers of the dermis is disturbed. The phrase “the structure of the bundles of collagenous fibers of the dermis is impaired” means that the structure of the bundles of collagenic fibers cannot be maintained in their normal form. For example, it includes the following: a bundle of collagen fibers becomes thin and weak, the amount of collagen fibers becomes small, collagen fibers are broken or there are not enough bundles of collagen fibers. Further, the phrase “normalizing the structure of the skin” also includes the regeneration of the damaged area of the skin. It also includes the restoration of the damaged area and the acceleration of the closure of the damaged area with regenerated tissue, which is able to prevent infection through the damaged area and restore the protective mechanism and respiratory function of the skin. Here, the phrase "skin defect" refers to a damaged state of the skin structure, where, for example, skin roughness, wrinkles and wounds have formed. A topical skin preparation of the present invention is particularly suitable for wound healing. Further, the phrase “normalizing the structure of the skin” also implies that the activity of keratinocytes to increase the ability of fibroblast to produce collagen is enhanced.

A topical skin preparation of the present invention can be used without any specific limitation as long as it is applied externally to the skin. Examples of a topical preparation include cosmetics containing pseudo-drugs, topical skin preparations, and topically applied dermatological compositions. Of these, it is particularly preferable to apply cosmetics and skin preparations for external use. For example, cosmetics are preferred to improve wrinkles and slight skin roughness. In particular, cosmetics are preferably used to restore bundles of collagenic fibers of the dermis in a disorder caused by aging under the influence of light, and to regenerate the structure of bundles of collagenic fibers of the dermis damaged by injury, or when severely damaged by the action of light. In contrast, skin preparations for external use are preferable for creating an improvement in severe skin roughness and for wound healing.

The dosage form of a topical skin preparation of the present invention is not specifically limited and may be any of the dosage forms that can be selected from traditional topical skin preparations, depending on the intended use. Preferred examples of dosage forms of cosmetics include a dosage form commonly used for basic skin care, such as lotion, lotion, essence, cream and tampon. In addition, preferred examples of dosage forms for topical application to the skin include ointment, lotion, cream, lotion and essence.

The content of the flavanone derivative and / or its salt in the preparation for local application to the skin of the present invention, which can be determined depending on the application, purpose and method of administration of the preparation for local application to the skin, is preferably 1 × 10 ^-6 % by weight and more preferably 1 × 10 ^-4 % by weight as a lower limit. The upper limit is preferably 1% by weight, and more preferably 0.1% by weight.

Further, especially in the case of topical preparations for skin application for wound healing, the content of the flavanone derivative and / or its salt may preferably be from 1 × 10 ^-6 M and more preferably 1 × 10 ^-4 M as the lower limit and preferably 1 M, and more preferably 0.1 M, as an upper limit.

Further, when a plant extract containing a flavanone derivative and / or its salt is contained in a topical skin preparation of the present invention, it is preferable to use an extract with an increased concentration of the flavanone derivative and / or its salt by purification of the flavanone derivative and / or salt. The preparation for local application to the skin of the present invention does not include the preparation for local application to the skin, used to correct bundles of collagenic fibers of the dermis, containing crude Hypericum erectum extract and not containing an additional derivative of flavanone, and / or its salt, other than the derivative originally included into the extract. The plant extract that can be used in the topical skin preparation of the present invention can preferably be an extract with an increased concentration of farrerol obtained by purification of the plant extract of Hypericum erectum. The concentration of the flavanone derivative and / or its salt in the plant extract is from 10 ^-6 to 0.1% by weight, and more preferably from 10 ^-5 to 0.01% by weight. In the case of using such a plant extract, the content of the plant extract in the preparation for topical application to the skin is preferably from 0.001 to 10% by weight and, more preferably, from 0.01 to 1% by weight. Here, the content of the plant extract is synonymous with its content in the product secreted by the solvent.

A topical skin preparation of the present invention may contain optional components commonly used in a topical skin preparation, as well as essential components. Preferred examples of such an optional component include: oils / waxes such as Australian nut oil, avocado oil, grain oil, olive oil, rapeseed oil, sesame oil, castor oil, sunflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, reconstituted coconut oil, reconstituted oil, vegetable wax, hardened castor oil, beeswax, candelilla wax, carnauba wax, thuja wax, animal wax, reconstituted animal wax, about its approved lanolin and jojoba wax; hydrocarbons such as liquid paraffin, squalane, pristan, ozokerite, paraffin, ceresin, petroleum jelly and microcrystalline wax; higher fatty acids such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenovub acid and undecylenic acid; higher alcohols, such as cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenic alcohol, octyldodecanol, myristyl alcohol, acetetearyl alcohol; synthetic ester oils such as tsetilizooktanoat, isopropyl myristate, geksildetsilizostearat, diisopropyl adipate, di-2-ethylhexylsebacate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentilglikoldikarat, di-2-heptylundecanoate glycerin, tri-2-ethylhexanoate, glycerol tri Trimethylolpropane 2-ethylhexanoate, trimethylolpropane triisostearate and pentaerythrol tetra-2-ethylhexanoate; silicon oil, such as chain polysiloxanes, such as dimethylpolysiloxane, methylphenylpolysiloxane and diphenylpolysiloxane; cyclic polysiloxanes such as octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane and dodecamethylcyclohexanesiloxane; modified polysiloxanes, such as amino-modified polysiloxane, polyester modified polysiloxane, alkyl-modified polysiloxane and fluorine-modified polysiloxane; anionic surfactants such as fatty acid soaps (such as sodium laurate and sodium palmitate), potassium lauryl sulfate and triethanolamine alkyl sulfate ester; cationic surfactants such as trimethylammonium stearyl chloride, benzalkonium chloride and laurylamine oxide; amphoteric surfactants such as imidazoline-based amphoteric surfactants (such as 1-carboxyethyloxy-2-cocoyl-2-imidazolinium hydroxide disodium salt), betaine-based surfactants (such as alkyl betaine, amide betaine and sulfobetaine) and acylmethyl taurine; non-ionic surfactants such as sorbitol fatty acid esters (such as sorbitol monostearate and sorbitol sesquioleate), glycerol fatty acid esters (such as glycerol monostearate), propylene glycol fatty acid esters (such as propylene glycol monostearate), castor oil derivatives, glycerol ethers, derivatives POE fatty acid esters of sorbitol (such as POE mono-oleate of sorbitol and polyoxyethylene sorbitan monostearate), POE fatty acid esters of sorbitol (such as POE-sorbitol monolaurate), POE fatty acid ester glycerol (such as glycerol POE monoisostearate), POE fatty acid esters (such as polyethylene glycol monooleate and POE distearate), POE alkyl ethers (such as POE2 octyldodecyl ether), POE alkyl phenyl ethers (such as POE nonylphenyl ether), pluron types, POE / POP alkyl ethers (such as POE / POP2 decyl tetradecyl ether), tetron types, POE castor oil / cured castor oil derivatives (such as POE castor oil and POE cured oil castor oil), ca fatty acid ester Arosa alkylglycosides; polybasic alcohols such as polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, mannitol, propylene glycol, dipropylene glycol, diglycerin, isopernglycol, 1,2-pentanediol, 2,4-hexanediol, 1,2-hexanediol and 1 2-octanediol; moisturizing components such as sodium pyrrolidone carboxylate, sodium lactate and sodium lactate; finely divided inclusions, such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicon anhydride (silicon oxide), aluminum oxide and barium sulfate, whose surfaces can be treated; inorganic pigments such as red iron oxide, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine blue, iron blue, titanium oxide and zinc oxide, whose surfaces can be treated; pearlescent reagents such as mica titanium, fish scale foil and bismuth oxochloride, whose surfaces can be treated; organic dyes such as Red No. 220, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201 and Red No. 204, which may be varnished; finely divided organic inclusions, such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomer; a para-aminobenzoate-based ultraviolet light absorbent; Anthranilate-based UV light absorbent; salicylate-based ultraviolet light absorbent; cinnamate-based ultraviolet light absorbent; benzophenone-based ultraviolet light absorbent; sugar based ultraviolet light absorbent; UV absorbers such as 2- (2'-hydroxy-5'-tert-octylphenyl) benzotriazole and 4-methoxy-4'-tert-butyldibenzoylmethane; lower alcohols such as ethanol and isopropanol; vitamins such as vitamin A or derivatives thereof; B-type vitamins, such as vitamin B ₆ hydrochloride, vitamin B ₆ tripalmitate, vitamin B ₆ dioctanoate, vitamin B ₂ or derivatives thereof, vitamin B _12, and vitamin B ₁₅ or derivatives thereof; E-type vitamins such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate, types of vitamin D, vitamin H, pantothenic acid, panthein, and pyrroloquinolinequinone; and antibacterial agents such as phenoxyethanol.

Of these, the optional component contained in topical skin preparations for accelerating wound healing is particularly preferably any of the components effective in regenerating skin tissue damaged by a wound. More specifically, an optical component, an antibiotic to prevent microbial infection, such as penicillin, fradiomycin, tetracycline, or a salt thereof, a sterilizer such as acrinol or isodine, or the like, may be particularly preferred. Alternatively, a scar suppressant, such as tranilast, is also preferred. The content of any of these components, effective for the regeneration of skin tissue, is preferably from 0.1 to 10% by weight.

A topical skin preparation of the present invention can be prepared by processing a flavanone derivative and / or its salt and optical component in a conventional manner.

In addition, the use of a topical skin preparation of the present invention can be modified depending on the application, dosage forms, place of application, symptom, and so on.

(C) A method for enhancing the action of keratinocytes to increase the ability of fibroblast to produce collagen

A characteristic feature of the method of enhancing the action of keratinocytes to increase the ability of fibroblasts to produce collagen of the present invention (hereinafter also referred to as the "method of the present invention") is the introduction of a flavanone derivative represented by the general formula (1) and / or its salt, in the presence of both keratinocytes and fibroblasts.

The flavanone derivative and / or its salt are used in the method of the present invention, following the description (A) above. The flavanone derivative and / or its salt are active in increasing the ability of fibroblast to produce collagen by acting on keratinocytes.

The meaning of the term “collagen” conceptually includes both collagen and collagen, which is the precursor of collagen.

The phrase "in the presence of both keratinocytes and fibroblasts" refers to a condition in which these cells coexist with each other in vitro or in vivo. For example, in vitro, this condition can be obtained by co-cultivation of these cells. In contrast, in vivo, such a condition can be found in the skin containing the epidermis and dermis.

The introduction of the flavanone derivative and / or its salt can be carried out, for example, by mixing the flavanone derivative and / or its salt with the culture medium for co-culturing keratinocytes and fibroblasts. In this case, the amount (dosage) of the mixture is preferably 10 ⁻⁸ M and, more preferably, 10 ⁻⁷ M as the lower limit and preferably 10 ⁻³ M and, more preferably, 10 ⁻⁵ M as the upper limit in terms of concentration in the environment.

Further, the in vivo administration method may be transdermal administration. The transdermal administration method is not particularly limited since it is commonly used, but a method for applying the preparation for topical application to the skin containing the flavanone derivative and / or its salt is preferred. A preparation for topical application to the skin, which can be used in the method of the present invention, is a preparation for topical application to the skin of the present invention, as described above, and a preferred embodiment of its manufacture is also carried out as described above.

(D) Screening Method of the Present Invention

A characteristic feature of the screening method of the present invention is that normal animal fibroblasts are cultured in the presence of a test substance, a collagen medium solution is added to the resulting culture to produce a collagen gel, fibroblasts in a collagen gel are cultured in the presence of a test substance, then a damaged area is formed in the collagen gel and fibroblasts are cultured in such a way that a collagen gel with a damaged area coexists with the test substance, the degree of olneniya or restore the damaged area is observed during cultivation.

The normal animal fibroblast used in the screening method of the present invention is not specifically limited if it is commonly used in a study. Preferred examples of the animal include mouse, rat, rabbit, and human. Of these, humans are particularly preferred since there are slight differences among animal samples. Normal fibroblasts are non-cancerous cells that have the ability to form an ordered collagen gel. Such normal fibroblasts can be obtained from the mucous membrane of the mouth, skin, or the like, or their primary culture can be obtained and applied. Alternatively, the investigated cell line culture is commercially available and such a commercial product can be used. A commercial product is preferred, for example, a product obtained as "Normal Human Skin Fibroblast (Primary Culture)" manufactured by Sanko Junyaku Co., Ltd., and "Normal Human Fibroblast" manufactured by Kurabo Co., Ltd.

Note that, in the future, normal fibroblasts of these animals are also briefly referred to as “fibroblasts”.

Preferably, the fibroblasts are pre-cultured in advance to the appropriate number of cells and then subjected to subsequent cultivation. Pre-cultivation can be carried out under general culture conditions for cell culture, for example, where a culture medium such as DMEM (manufactured by GIBCO) is added and applied with 10% FBS and the cells are then incubated at 37 ° C under a stream of 5% carbon dioxide gas to as long as cell growth is observed up to 60 to 90% of the completeness of filling. The cell cluster obtained by pre-cultivation is preferably partitioned into individual cells in the presence of 0.01-0.1% trypsin, to obtain a cell suspension. In this case, the presence of ethylenediaminetetraacetate with trypsin is more preferred.

For culturing fibroblasts in the presence of a test substance, the method of adding a test substance is not particularly limited. The test substance can be added to the medium in advance, added at the same time as the cells are seeded, or added after the cells are seeded and then cultured for an appropriate time. Preferably, the method may be a method in which cells are first seeded on a plate and then cultured in a medium free of the test substance, followed by replacing the medium with a nutrient medium containing the test substance. The added test substance may have any concentration. Preferably, the cultivation is carried out using media containing various concentrations of the test substance to test the effective amount of the test substance. In addition, the number of fibroblast cells used in culture is not specifically limited and can be set according to the size of the culture plate. For example, the number of cells can preferably be set from 10 ³ / cm ² to 10 ⁵ / cm ² . The cultivation can be carried out under normal conditions in a manner similar to pre-cultivation. The culture holding time is not specifically limited, as long as the number of cells can increase to an appropriate number, so that it can, for example, be from about 3 to 5 days, stereotypically. During the period, if required, the medium may be replaced by a nutrient medium.

More specifically, for example, a cell suspension obtained by rupturing pre-cultured fibroblasts into individual cells was seeded onto tablets and then cultured for about 24 hours, followed by the addition of a test substance solution at various concentrations. The cultivation was preferably carried out for 3 to 5 days.

A collagen gel was then prepared by adding a collagen medium solution to the fibroblast culture obtained as described above. The amount of collagen medium solution added and the collagen concentration in the collagen medium solution are not specifically limited as long as they are within the range acceptable for fibroblast dispersion in the collagen gel. The applied collagen medium solution can be, for example, a solution obtained by mixing from 0.3 to 0.8% by weight solution of type-I collagen, DMEM medium, 100 to 300 mM HEPES, 2-4% by weight of bicarbonate solution sodium, aqueous 0.05-0.15 n sodium hydroxide solution, FBS, and water in a ratio of about 4: 4: 2: 2: 1: 2: 3. The amount of the added collagen medium solution can be determined so that, for example, the ratio of the collagen medium solution to the fibroblast culture is from 8 to 10 parts by weight to 1-2 parts by weight.

Then the fibroblasts in the resulting collagen gel were cultured in the presence of the test substance. In other words, the collagen gel obtained as described above was placed in a medium containing a test substance, thereby forming a suspension of fibroblast culture in a collagen gel.

The concentration of the test substance in the medium used in the culture may correspond to the concentration of the medium used in the fibroblast culture in the presence of the test substance. The culture holding time can be controlled depending on the degree of recovery of the collagen gel. More specifically, the cultivation is preferably carried out until about 1/5 to 1/10 of the restoration of the size of the gel was achieved relative to its size at the time of preparation of the gel with the addition of a collagen solution. The culture holding time can typically be 3 to 5 days.

Then created a damaged area in the restored collagen gel. The damaged area was a segment where a hole was punched in part of the collagen and kept hollow. The appearance and size of the damaged area is not specifically limited as long as it is suitable for observing a change in appearance. For example, the central part of the restored collagen gel can be perforated to a round shape, which leads to a notch of a toroidal shape. The punching technique can use, for example, a disposable biopsy punch with a diameter of 1 to 5 mm.

A method for producing such a damaged collagen gel model is first described in Roman O'Leary, Mark Rerek, and Edward John Wood, Biol. Pharm. Bull., 27 (2), 266-270 (2004).

Then a collagen gel equipped with a damaged area was placed in the medium of the test substance, and then the fibroblasts were cultured in a collagen gel. The tablet used for cultivation may be selected depending on the size of the resulting collagen gel. For example, if the collagen gel has a diameter of about 2 to 7 mm, a 6-well plate may be selected. In addition, the collagen gel is preferably fixed at the bottom of the well. Fixation of the collagen gel can be carried out by, for example, dropping a collagen solution on the central part of the well and incubating the collagen gel on it for 15 minutes at 37 ° C. The concentration of the test substance in the medium may correspond to the concentration in the medium used in the cultivation of the aforementioned fibroblasts in the presence of the test substance. The cultivation conditions used may also be conventional conditions, just as in the case of the above cultivation conditions. The cultivation period (i.e. the observation period), without limitation, typically ranges from 5 to 20 days.

During the cultivation, as described above, the degree of filling or restoration of the damaged area of the collagen gel was observed. By recording the observation results, the action of the test substance in the direction of collagen reconstruction can be determined. For example, the appearance of the damaged area can be objectively recorded using photographing. Optionally, these photographs can be compared and measured by converting them into a digital image. More specifically, when the degree of filling or restoration of the aforementioned damaged area in the presence of the test substance is greater than the degree of recovery in the absence of the test substance, it can be determined that the aforementioned test substance has the effect of accelerating wound healing. In addition, the degree of filling or restoration can be examined, so that an estimate, or the like, is used to replace the degree of filling or restoration with a digital value. For example, when the volume of the aforementioned damaged area in the presence of the test substance is 85% or less and preferably 50% or less of the volume in the absence of the test substance, it can be determined that the aforementioned test substance has the effect of accelerating wound healing.

Thus, the effect of accelerating the wound healing of a test substance can be investigated by such a method, it becomes possible to remove any substance that only accelerates fibroblast migration and does not enhance collagen production by fibroblasts or any substance that only enhances collagen production by fibroblasts and does not accelerate migration (movement) fibroblasts. Therefore, any substance effective as a wound healing agent can be more effectively found.

EXAMPLES

Example 1

The corrective effect of farrerol on bundles of collagenic fibers of the dermis was confirmed by an in vitro experiment. In other words, given the fact that when fibroblasts are cultured in a collagen gel in a medium containing heparin, collagen fibers obtained from cells are damaged and strong bundles of collagen fibers cannot be obtained, the degree of recovery from damage to collagen fiber was investigated by adding farrerol to coexist with a heparin containing system. The techniques will be described below.

<Applied cells and reagents>

Normal human fibroblasts (Kurabo)

DMEM, with 10% FBS (GIBCO)

WST-8/1-Methyl-PMS (Kit-8 Cell Counting Kit: Dojindo laboratories)

Farrerol was dissolved in dimethyl sulfoxide (DMSO) and then used.

The toxicity of farrerol was investigated in an MTT study and then a dose of farrerol was added to the heparin-containing system.

<Protocol>

1. Fibroblasts grew to 80% completeness, dispersed in 0.05% Trypsin / EDTA medium. The dispersant was plated on a 96 well plate at a concentration of 3 × 10 ³ cells / well.

2. After 24 hours, the specified concentration farrerol was added to the corresponding wells in a volume of 10 ml / well (n = 8).

3. WST-8/1-Methyl-PMS was added to the corresponding wells in a volume of 10 ml / well 24 hours, 48 hours and 72 hours after the addition of farrerol, then incubated at 37 ° C for 4 hours.

4. The optical density was measured at a working wavelength of 450 nm and a reference wavelength of 650 nm.

<Results>

Cytotoxicity could not be confirmed as significant at any of the working concentrations. In contrast, it was confirmed that the addition of farrerol can enhance cell proliferation. In particular, cell proliferation was enhanced at concentrations of farrerol 10 ^-6 ml / ml-DMEM, 5 × 10 ^-7 ml / ml-DMEM, and 10 ^-7 ml / ml-DMEM, so a subsequent collagen production test was carried out using these three concentrations.

Based on these results, the effect of farrerol on enhancing collagen production using a system containing heparin was investigated.

<Applied cells and reagents>

Normal human fibroblasts (Kurabo)

HEPARIN (SIGMA: H-3149)

DMEM (GIBCO)

Fbs

<Protocol>

1. Fibroblasts were grown to completion in 10% FBS medium and then dispersed by trypsin treatment followed by centrifugation.

2. The supernatant was removed and then the fibroblasts were resuspended in DMEM, with 10% FBS, followed by plating of fibroblasts on a 12-well plate (4.5 cm ² / well) at a concentration of 3 × 10 ³ cells / cm ² .

3. 4 hours after plating of the cells, the DMEM supernatant was removed and replaced with DMEM medium with 0.4% FBS followed by 72 hours incubation under conditions to prevent cell proliferation (37 ° C, 5% CO ₂ ).

4. DMEM with 0.4% FBS was removed, then replaced with DMEM medium, with 10% FBS to complete the cessation of cell proliferation. In this technique, the culture conditions were 10% FBS-DMEM, 0.1% heparin / 10% FBS-DMEM, and 1.0% heparin / 10% FBS-DMEM (each n = 3) to study the effect of heparin. Next, prepared media with added farrerol at concentrations of 10 ^-6 ml / ml-DMEM, 5 × 10 ^-7 ml / ml-DMEM and 10 ^-7 ml / ml-DMEM, respectively.

<Results>

The results are presented in a graphical diagram in FIG. 1. Diagram 1 represents the effect of cell proliferation when each of the concentrations of the farrerol solution is added to the medium in the absence of heparin and Figure 2 represents the effect in the presence of heparin. Any of the diagrams shows that in the group with the addition of farrerol there is a tendency to enhance cell proliferation compared to the control group added with DMSO. Thus, it was found that farrerol has the effect of enhancing the growth of fibroblasts.

Example 2

The experiment above confirms that farrerol has the effect of enhancing cell proliferation. In addition, to confirm the effect of farrerol on the ability of fibroblasts to move, the “wound healing model" obtained by perforating a three-dimensional collagen gel with a disposable biopsy punch was used in comparison between the group with the addition of farrerol and the group without the addition of farrerol, in relation to the behavior of fibroblasts in the region wounds.

<Applied cells and reagents>

Normal human fibroblasts (Kurabo)

DMEM, with 10% FBS (GIBCO)

A solution of collagen medium (0.5% type-I collagen: 5 × DMEM: 200 mm HEPES: 2.2% NaHCO ₃ : 0.1 n NaOH: FBS: water was mixed in a ratio of 4: 4: 2: 2: 1: 2: 3)

Disposable Biopsy Hammer (3.0 mm in tip diameter)

<Protocol>

1) The fibroblast culture obtained according to the protocol 4 of Example 1 and the collagen medium solution were mixed together at a ratio of 1: 9 (by volume) and then the suspension was cultured in 10% FBS medium for 1 week.

2) When the size of the collagen gel decreased from the size at the time when it was obtained by adding a solution of collagen medium to a size of about 1 / 5-1 / 10, the central part of the gel was perforated with a disposable biopsy puncher with a diameter of 3 mm.

3) A 6-well plate was prepared and 15 μl of collagen solution was then dropped onto the center of the well. Then a perforated three-dimensional collagen gel was placed in the well and incubated for 10 minutes (37 ° C, 5% CO ₂ ).

4) When the collagen droplets cured and the three-dimensional collagen gel was attached to the bottom of the well, 10% FBS-DMEM, 0.1% heparin / 10% FBS-DMEM, and 1.0% heparin / 10% FBS-DMEM were added in a volume of 3 ml / well.

5) Next, a group containing 10 ^-6 ml / ml-DMEM of farrerol was added to each culture, conditions were obtained (4).

<Results>

Fibroblast behavior was observed 24 hours after receiving the “wound healing model”. In FIG. 2 shows photographs of fibroblasts taken for a wound region perforated with a disposable biopsy punch. With respect to the incomplete medium of 10% FBS-DMEM, there was a tendency to a longer distance of fibroblast movement and more active cellular movement in the group with added farrerol, compared with the group without added farrerol. In contrast, with respect to the medium 0.1% heparin / 10% FBS-DMEM and 1.0% heparin / 10% FBS-DMEM, there is no difference in cell movement between the group with added farrerol and the group without added farrerol, the movement in each of they are prevented by the action of heparin.

Therefore, the results show that farrerol does not inhibit the effect of heparin on the ability of fibroblasts to move, but directly affects fibroblasts, increasing the ability of cells to move.

Example 3

The following methods were used to investigate whether farrerol is effective in preventing the inhibitory effect of added heparin on the construction of bundles of collagen fibers. The results are presented in FIG. 3. It was found that farrerol prevents the inhibitory effect of heparin on the construction of bundles of collagen fibers and has a corrective effect on bundles of collagen fibers of the dermis.

<Protocol>

A collagen solution was obtained by preparing a mixture of 0.5% collagen type I: 5 × DMEM: 200 mm HEPES: 2.2% NaHCO ₃ : 0.1 n NaOH: FBS: water in a ratio of 4: 4: 2: 2: 1: 2: 3 (carried out with cooling).

↓

The lower layer of the collagen solution was obtained by mixing a collagen solution with water in a ratio of 9 to 1.

↓

The lower layer of collagen solution was dispensed into the corresponding wells of a 48-well plate in a volume of 100 μl / well.

↓

Curing was carried out in a CO ₂ incubator (about 15 minutes).

↓

NHDF was prepared according to standard procedures.

(The cells were separated from each other by a cell sieve).

↓

The number of cells was measured (Trypan blue) and then brought to a concentration of 1 × 10 ⁵ cells / ml.

↓

The collagen solution was mixed with the cell suspension in a ratio of 9 to 1 (by volume) and then distributed into the corresponding wells in a volume of 300 μl / well.

To prevent uneven cell dispersion, the operation was performed with intermittent stirring (1 × 10 ⁴ cells / ml in the end).

↓

Cells were left to stand for 4 hours in a CO ₂ incubator.

↓

The gel was separated from the inner wall of the well by an injection needle, without stopping the restoration of the gel.

↓

Addition of 1.8% Heparin / 10% FBS-DMEM medium was performed in a volume of 500 μl / well (final concentration of 1% heparin per well).

↓

Farrerol (dissolved in DMSO) was added in a volume of 0.9 μl / well (1000-fold dilution).

↓

Incubation was performed in a CO ₂ incubator (5 days).

↓

Got a sample for observing SEM.

Example 4

According to the composition described below, received cosmetic products (essences), which are external compositions for the skin of the present invention. A, B and C were heated to 80 ° C, B was gradually added to A with stirring to emulsify, and then C was added to neutralize, followed by stirring under cooling. The result was an essence 1. A similar operation was carried out to obtain Comparative Example 1, in which the farrerol in essence 1 was replaced by ethanol.

Table 1 Components % by weight BUT Squalane 5 POE Behene Ether (20) one Began alcohol 2 Sorbitol Sesquistearate 1,5 Behenic Acid 0.5 AT 1,3-butanediol 8 1,2-hexanediol four Phenoxyethanol 0.5 "Carbopol 1382" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) "Pemulen TR-1" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) Water 57.1 0.1% solution of farrerol in ethanol 0.1 FROM Potassium hydroxide 0.1 Water twenty In total one hundred

<Test Example 1>

Using two groups of 5 patients, a total of 10 participants (aged 40 to 50 years) were subjected for 8 weeks to the test for applying the essence 1, obtained as described above, and cosmetics of Comparative Example 1. These cosmetics were used twice: every morning and at night every day; received exact copies of the corners of the eyes before and after the test application. When illuminated with a light with an angle of inclination of 45 ° C, the shadows formed by wrinkles were transferred to the pictures from a microscope and then digitized to obtain the ratio of the area of the shadows in the field of view, followed by their average value for the group. The results are shown in table 2. Thus, it was found that cosmetics (essences), which are a preparation for topical application to the skin of the present invention, have an excellent effect on improving the condition of wrinkles.

Sample The average ratio of shading areas (%) Before the test After the test Essence 1 6.8 ± 2.3 3.3 ± 0.9 Comparative Example 1 7.1 ± 2.8 5.7 ± 1.6

Example 5

<Test Example 2>

Essence 2, which is a preparation for topical application to the skin of the present invention, was obtained according to the following composition in a manner analogous to the method for essence 1. It was examined by the method described in Test Example 1. Therefore, the effect was confirmed that the ratio of wrinkle areas varies from 8.2 ± 3.2% before the test to 3.9 + 1.1% after the test.

Table 3 Components % by weight BUT Squalane 5 Behene ether POE (20) one Began alcohol 2 Sorbitol Sesquistearate 1,5 Behenic Acid 1,5 AT 0.5 1,3-butanediol 8 1,2-hexanediol four Phenoxyethanol 0.5 "Carbopol 1382" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) "Pemulen TR-1" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) Water 57.1 0.001% solution of farrerol in ethanol 0.1 FROM Potassium hydroxide 0.1 Water twenty In total one hundred

Example 6

Using the "model of defective skin tissue with a wound" in which the damaged area was obtained by perforating the collagen gel, the effectiveness of farrerol with respect to enhancing tissue regeneration of the damaged area was studied. The techniques will be described below. The results are presented in FIG. 4. The figure found that the decrease in the size of the perforated hole is obviously accelerated at a final concentration of ^10-5 M or 10 ^-6 M farrerol, but the acceleration of recovery becomes unclear at a concentration of 10 ^-7 M. Therefore, it was found that the lower limit of the concentration of farrerol preferably 10 ^-6 M in the preparation for topical application to the skin of the present invention.

1) Normal human fibroblasts grew to 80% completeness, dispersed in 0.05% Trypsin / EDTA medium. Fibroblasts were seeded at a cell density of 3 × 10 ⁵ cells / 25 cm ² .

2) After 24 hours, the medium was replaced with medium supplemented with farrerol (10 ^-5 M, 10 ^-6 M or 10 ^-7 M in final concentration) and DMSO, followed by cultivation for 4 days.

3) A collagen gel was prepared by adding a collagen medium solution to the culture obtained by culturing under each condition in a manner similar to that of Example 2. The resulting collagen gel was then cultured in suspension for 1 week in the same medium as the culture medium of the culture conditions according to (2) . Fibroblasts cultured in a medium with 10 ^-5 M, 10 ^-6 M and 10 ^-7 M farrerol in (2), after preparation of the collagen gel, were cultured in a medium with 10 ^-5 M, 10 ^-6 M and 10 ^-7 M farrerol, respectively .

4) When the size of the collagen gel was reduced compared with the size at the time of receipt by adding a solution of collagen medium to a size of about 1 / 5-1 / 10, the central part of the gel was perforated in the shape of a toroid with a disposable biopsy puncher with a diameter of 3 mm. The result was a "wound model".

5) A 6-well plate and 5 μl of collagen were prepared, then dripped onto the center of the well. Then the collagen gel of the wound model was distributed there and incubated at 37 ° C for 15 minutes.

6) When the collagen added dropwise solidified and the gel was attached to the bottom of the well, 3 ml of medium was added with the same state as state (3), followed by culturing as a “model of defective skin tissue with a wound”. Then, the process of closing the wound area of the collagen gel was observed visually.

Example 7

According to the composition described below, cosmetic preparations (essences) were prepared, which are a preparation for topical application to the skin of the present invention. A, B and C were heated to 80 ° C, B was gradually added to A with stirring to emulsify, then C was added to neutralize, followed by stirring under cooling. The result was an essence 3. A similar operation was performed to obtain Comparative Example 2, in which the farrerol in essence 3 was replaced by ethanol.

Table 4 Components % by weight BUT Squalane 5 Behene ether POE (20) one Began alcohol 2 Sorbitol Sesquistearate 1,5 Behenic Acid 0.5 AT 1,3-butanediol 8 1,2-hexanediol four Phenoxyethanol 0.5 "Carbopol 1382" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) "Pemulen TR-1" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) Water 57.1 Material extracted with 80% aqueous ethanol in Production Example 1 0.1 FROM Potassium hydroxide 0.1 Water twenty In total one hundred

<Test Example 3>

Using essence 3 and Comparative Example 2, the effect of accelerating tissue regeneration of the damaged area with a wound was studied on the participants of the experiment. The stripped stamp was placed on the middle part of the upper part of the hand of the participant of the experiment and the epidermis was removed with a scalpel to obtain three fragments of 1 mm × 1 mm. Essence 3 was applied to one fragment once a day for 5 days from the day the epidermis was removed. Comparative Example 2 was applied to another fragment once a day for 5 days from the day the epidermis was removed, and one remaining fragment was not treated. The observation was carried out 1 week and 2 weeks after application to determine the degree of tissue regeneration in the damaged area. The results are presented in table 5. Therefore, the essence 3, a preparation for topical application to the skin of the present invention, was found to have an excellent effect of accelerating tissue regeneration of the damaged area. In addition, scar formation was not observed at all.

Table 5 Sample Observation of the result after 1 week Observation of the result after 2 weeks Essence 1 Most damaged areas recovered No differences with normal areas. Comparative Example 1 Crust present and partial defect remains Most damaged areas recovered Raw Crust present and partial defect remains Most damaged areas recovered

Example 8

<Test Example 4>

According to the composition described below, a topical skin preparation 1 of the present invention was prepared in a manner analogous to the method of Example 7. The same assessment was carried out as in Test Example 3. As a result, the preparation showed an excellent effect of accelerating the regeneration of defective skin tissue even with simultaneous presence with an antibiotic compound.

Table 6 Component % by weight BUT Squalane 5 Behene ether POE (20) one Began alcohol 2 Sorbitol Sesquistearate 1,5 Behenic Acid 0.5 AT 1,3-butanediol 8 1,2-hexanediol four Phenoxyethanol 0.5 "Carbopol 1382" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) "Pemulen TR-1" 0.1 (Alkyl-modified carboxyvinyl polymer manufactured by Goodrich Corporation) Water 56.1 Fradiomycin Sulfate one Material extracted with 80% aqueous ethanol in Production Example 1 0.1 FROM Potassium hydroxide 0.1 Water twenty In total one hundred

Example 9

The effect of farrerol on keratinocytes was investigated according to the following methods.

<Experiment 1: assessment of the cytotoxicity of farrerol on keratinocytes>

[Applied cells and reagents]

Normal human epidermal keratinocytes (manufactured by Kurabo Co., Ltd.)

Humedia KG2 (manufactured by GIBCO Co., Ltd.)

WST-8/1-Methyl-PMS (Kit-8 Cell Counting Kit: Dojindo laboratories)

[Sample preparation]

A farrerol sample was prepared by diluting DMSO 10 ^-2 M farrerol dissolved in DMSO to adjust its concentration.

[Protocol]

1) Normal human epidermal keratinocytes grew to 80% fullness, dispersed in 0.05% Trypsin / EDTA medium. The dispersion was plated on a 96 well plate at a concentration of 3 × 10 ³ cells / well and 6 × 10 ³ cells / well.

2) After 24 hours, a sample of farrerol with the established concentration was added to the corresponding wells in a volume of 100 μl / well (n = 8).

3) WST-8/1-Methyl-PMS was added to the corresponding wells in a volume of 10 μl / well after 24 hours and 48 hours after adding a sample of farrerol, and then incubated at 37 ° C for 4 hours.

4) The optical density (OD value) was measured at a working wavelength of 450 nm and a reference wavelength of 650 nm.

<Results>

The evaluation results with MTT are presented in the graphical diagram in FIG. 5. It shows that the OD value increases with the concentration of farrerol. The OD value, for 3 × 10 ³ cells / well after 24 hours, 10 ^-5 M farrerol, shows a value two or more times higher than the control value in the case of DMSO. After 48 hours, a value 1.7 times higher than the value in the case of DMSO is also recorded. However, the number of cells was small and their OD value tends to be low, so reproducibility of the results is confirmed by the use of a double row of seeded cells, 6 × 10 ³ cells / well. As a result, an almost equal result can be obtained.

On the other hand, visual assessment does not give the impression that cell proliferation in the group with added farrerol is accelerated compared with the group with added DMSO.

<Experiment 2: Effect of farrerol on the proliferation of keratinocyte cells (direct study)>

[Applied cells and reagents]

Normal human epidermal keratinocytes (manufactured by Kurabo Co., Ltd.)

Humedia KG2 (manufactured by GIBCO Co., Ltd.)

[Protocol]

1) An environment of normal keratinocytes of the human epidermis was obtained at a cell density of 6 × 10 ³ cells / ml and then 1 ml aliquots of it were plated on a 96 well plate, respectively.

2) After 24 hours, the supernatant was removed from the respective wells and then 1 ml of medium with added sample of farrerol was added to each of the wells at a concentration determined by dilution with DMSO.

3) 24 hours, 48 hours and 72 hours after the addition of the sample, the cells were dispersed with 0.05% Trypsin / EDTA medium and their number was then measured with a blood cell counter. In this way, in Experiment 1, the score for WST-8 was used as an indicator of cell number. Since this method is provided for indirect estimation of the number of cells by acting on dehydrases in mitochondria, the present study directly measures the number of cells.

[Results]

Cell counting results are shown in FIG. 6. The diagram represents the cell proliferation rate when the number of cells in the control (group with the addition of DMSO) is defined as 100%. Compared to the control, the proliferation rate of cells tends to be inhibited when there is a concentration dependence. The cell proliferation rate in the 10 ^-5 M group with added farrerol was inhibited to 75.1 ± 5.2% after 24 hours. In addition, in relation to other concentrations, cell proliferation rates were maintained at 75.5 ± 5.8% at 10 ^-6 M and 77.1 ± 8.6% at 10 ^-7 M. After 48 hours, 10 ^-5 M the group with added farrerol showed a proliferation rate of about 71.9 ± 16.6%. However, at any concentration lower than the aforementioned concentration, almost the same proliferation rate was recorded as the rate in the control group. In addition, there was no change in cell appearance (Fig. 7). That is, the results of a direct assessment did not coincide with those indirect estimates of the number of cells in the case of WST-8. As a result, it was understood that farrerol could accelerate the reaction of the citric acid cycle in keratinocyte mitochondria. Thus, it is expected that the effect of NADH formed during the reaction of the citric acid cycle can also accelerate the subsequent reaction step, the oxidative phosphorylation reaction. As a result, it was shown that the generation of ATP, which provides a source of energy in the cell or the capacity of metabolic energy, can be accelerated.

<Experiment 3: Investigation of the indirect effect on fibroblasts, through epidermal keratinocytes>

[Summary]

To detect whether or not an interaction between epidermal keratinocytes and fibroblasts, or any effect of farrerol on fibroblasts in a co-culture, was evaluated, the amount of procollagen produced was evaluated. The method is based on the method described in Eming SA et al., Hum. Gene. Ther., 9 (4), 529-39 (1998).

[Applied cells and reagents]

Normal human epidermal keratinocytes (manufactured by Kurabo Co., Ltd.)

Humedia KG2 (manufactured by Kurabo Co., Ltd.)

Normal human fibroblasts (manufactured by Kurabo Co., Ltd.)

DMEM (manufactured by GIBCO Co., Ltd.)

PIP EIA Kit (manufactured by TAKARA BIO INC.)

[Protocol]

1) epidermal keratinocytes

Day 1: cells were plated on a 24-well plate at a cell density of 3 × 10 ⁴ cells / well.

Day 6: A plate washed with PBS and medium was then replaced with DMEM + 2% FBS, followed by the addition of farrerol (10 ^-6 M, 10 ^-7 M, 10 ^-8 M, DMSO).

Day 7: harvested culture supernatant.

2) Fibroblasts

Day 3: cells were plated on a 24-well plate at a cell density of 2.5 × 10 ⁴ cells / well.

Day 7: The plate was washed with PBS and then supernatant culture (900 μl) of epidermal keratinocytes was added, followed by cultivation for 48 hours.

Day 9: The plate was washed with PBS and serum-free DMEM and the medium was then replaced with 500 μl of serum-free DMEM, followed by culturing for 2 hours. The culture supernatant was collected and the amount of procollagen in the centrifuged supernatant (15,000 rpm, 5 s) was then measured by the method described below.

[Quantification of the amount of procollagen obtained]

1) After adding 100 μl of the standard antibody solution to each of the plate wells, the previously prepared PIP standard solution at each of the concentrations (320, 160, 80, 40, 20 and 10 μl / ml; the test substance was diluted with DMEM) and 20 μl of the test sample added to each well of two rows by means of a micropipette, followed by incubation at 37 ° C for 3 hours (first reaction).

2) The reaction solution was removed, and the wells were then washed with PBS medium four times. Then, 100 μl of substrate solution (TMBZ) was added to each well using an eight-pipette pipette, followed by incubation at room temperature (20-30 ° C) for 15 minutes (second reaction).

3) Then, 100 μl of stopping solution was added to each well in the order of adding a substrate solution to stop the reaction, and then to the mixed well.

4) The standard curve was obtained by plotting the values of optical density at a wavelength of 450 mm, and the optical density of the medium was taken as the zero level. Then the PIP concentration was read from the corresponding optical density of the test substance.

[Results]

The results are presented in FIG. The amount of procollagen increases significantly depending on the concentration of farrerol. On the other hand, the other system, which was evaluated by the method of adding farrerol during the cultivation of fibroblasts and then the supernatant of the epidermal keratinocyte culture was added, did not show a large change in the amount of procollagen obtained. From this fact it follows that the effect of farrerol on fibroblasts, which directly increases the production of collagen, may be negligible. Therefore, we can assume that farrerol acts on keratinocytes of the epidermis and keratinocytes of the epidermis accelerate the ability of fibroblasts to enhance collagen production. In this way, the ability of fibroblast to produce collagen can be changed by using keratinocytes in a co-culture system. So, for example, with respect to a three-dimensional skin culture or the like, the method is applicable to changing the properties of three-dimensional skin to the desired properties.

Example 10

A preparation containing farrerol for topical application to the skin (cosmetics: lotion) was prepared according to the composition described below. The recipe components were mixed and dissolved at room temperature to obtain a lotion. The amount of procollagen formed in the dermis of the skin can be increased by transdermal injection of the lotion into the skin of a mouse or human, which leads to an increase in the amount of collagen in the dermis. This is because keratinocytes in the skin are found together with fibroblasts.

Table 7 Components % by weight 1,3-butanediol 5 1,2-pentanediol 2 1,2-hexanediol 3 Glycerol 5 Phenoxyethanol 0.5 Hypericum erectum extract from Production Example 1 0.001 Water 89,499 In total one hundred

Example 11

<Preparation of test substance>

As a test substance for screening, Hypericum erectum extract was prepared. 10 L of an 80% aqueous ethanol solution was added to 1 kg of the dried aerial part of Hypericum erectum Thunberg (Guttiferae) and then refluxed for 4 hours with stirring. After cooling, the insoluble residue was removed by filtration and then concentrated under reduced pressure, followed by freeze drying. The result was 51 g of amorphous substance. This product was dispersed in water, placed in a DIAION HP20, washed with a stream of 5 L of water and 5 L of a 50% aqueous solution of ethanol, in that order, and washed with 99% ethanol. The obtained eluted fraction was fractionated. Then, the fractions thus obtained were further subjected to thin-layer chromatography and HPLC to control their content, with the aim of jointly combining the fractions, including the same individual substance. On the other hand, fractions comprising two or more components were further purified by re-chromatography. As a result, seven fractions containing one component were obtained.

<Screening Example>

Fractions containing one component obtained as described above were provided as test substances and the effect of each test substance on collagen production was then examined to find a substance with a wound healing effect. Special techniques will be described below.

Using the “wound model” provided by the damaged area formed by perforating a collagen gel containing animal fibroblasts, the degree of filling or restoration of the damaged area in the presence of the test substance can be observed. The techniques are as follows. The structure of the test substance of a single component with the highest degree of filling or restoration of the damaged area, compared with the rest of the tested substances, was studied by proton NMR, mass spectrometric analysis, ¹³ C-NMR and identified as farrerol. In this way, the yield of farrerol from the above extract was 3.2 mg. The content of farrerol in the product released by the solvent into the extract by the action of an 80% aqueous solution of ethanol is about 0.006% by weight.

Further, the final concentration of farrerol was adjusted to any of 10 ^-5 M, 10 ^-6 M and 10 ^-7 M, and then the degree of restoration of the damaged area was observed at each concentration. The results are presented in FIG. 9. From the figure it was found that the restoration of the perforated hole (damaged area) is undoubtedly accelerated at a final concentration of farrerol of 10 ^-5 M or 10 ^-6 M, but the acceleration of recovery at 10 ^-7 M is not clear. Therefore, the effect of farrerol to accelerate the building of collagen can create an effect of accelerating wound healing when at least 10 ^-6 M farrerol is used. Thus, according to the screening method of the present invention, an effective concentration of the test substance with the effect of accelerating wound healing can also be achieved.

<Methods>

1) Normal human fibroblasts were grown to 80% completeness upon preliminary cultivation, dispersed in 0.05% Trypsin / EDTA. Fibroblasts were then plated on a plate at a cell density of 3 × 10 ⁵ cells / 25 cm ² (1 × 10 ⁴ cells / cm ² ).

2) After culturing for 24 hours, the medium was replaced with medium supplemented with farrerol (10 ^-5 M, 10 ^-6 M or 10 ^-7 M at the final concentration) and DMSO, followed by cultivation for 4 days.

3) A collagen gel was obtained by adding a solution of collagen medium to the obtained culture in a manner analogous to the method of Example 3. Fibroblasts in a collagen gel were cultured using the medium used in (2). The concentration of the test substance in the medium corresponded to the concentration of the medium (2). That is, fibroblasts cultured in a medium with farrerol with final concentrations of 10 ^-5 M, 10 ^-6 M and 10 ^-7 M in (2) were cultured in a medium with farrerol with final concentrations of 10 ^-5 M, 10 ^-6 M and 10 ^-7 M, respectively, after preparation of the collagen gel.

4) After cultivation for 1 week, the size of the collagen gel was restored compared with the size at the time of receipt by adding a solution of collagen medium to a size of about 1 / 5-1 / 10. Then the central part of the collagen gel was perforated to obtain a toroidal shape of the notch by the action of a disposable biopsy puncher with a diameter of 3 mm. The result was a "wound model".

5) A 6-well plate and 5 μl of collagen solution were prepared, then dripped onto the center of the well. A collagen gel model of the wound was then established and incubated at 37 ° C for 15 minutes, allowing the collagen gel to attach to the bottom of the well.

6) Then 3 ml of the same medium as the medium in (2) was added, to each of the wells and fibroblasts in the above collagen gel were then cultured for 7 days. During cultivation, the process of closing the wound area of the collagen gel was observed visually.

INDUSTRIAL APPLICABILITY

The preparation for topical application to the skin of the present invention is applicable for cosmetics, skin therapy for topical application, and so on. In addition, the use of a topical skin preparation of the present invention may contribute to wound healing.

Further, with respect to a three-dimensional skin culture or the like, a method can be applied to enhance the ability of fibroblast to produce collagen to generate artificial skin by correcting the properties of three-dimensional skin to the desired properties.

A substance with an accelerating wound healing effect suitable for topical skin preparations can be tested using the screening method of the present invention.

Claims (8)

1. A composition for topical application to the skin, intended for wound healing and containing an effective amount of a flavanone derivative represented by the following general formula (1) and / or its salt:

general formula (1),
in which R1, R2, R3, R4, and R5 each independently represent a hydrogen atom or an alkyl group having 1-4 carbon atoms. 2. The composition according to claim 1, in which the flavanone derivative is a farrerol.

farrerol 3. The composition according to claim 1, in which the content of the derivative of flavanone and / or its salt is from 1 · 10 ^-6 to 1% by weight. 4. A composition for topical application to the skin, intended for wound healing and comprising an extract of Hypericum erectum, Guttiferae, which contains from 10 ^-6 to 0.1% by weight of the flavanone derivative represented by the following general formula (1) and / or its salt :

general formula (1),
in which R1, R2, R3, R4, and R5 each independently represent a hydrogen atom or an alkyl group having 1-4 carbon atoms. 5. A wound healing method comprising administering an effective amount of a flavanone derivative represented by the general formula (1) and / or its salt:

general formula (1),
in which R1, R2, R3, R4, and R5 each independently represent a hydrogen atom or an alkyl group having 1-4 carbon atoms. 6. The method according to claim 5, in which the flavanone derivative is a farrerol. 7. The method according to claim 5, in which the dosage of the derivative of flavanone and / or its salt is 10 ^-8 M or more. 8. A wound healing method comprising administering an extract of Hypericum erectum containing an effective amount of a flavanone derivative represented by the general formula (1) and / or its salt:

general formula (1),
in which R1, R2, R3, R4, and R5 each independently represent a hydrogen atom or an alkyl group having 1-4 carbon atoms.

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