JP7682666B2 - Nitrated protein degraders - Google Patents

Description

The present invention relates to a lactic acid bacteria fermentation product of rose petals, and more specifically to a nitrated protein decomposition agent and topical skin preparation that contain a fermentation product obtained by fermenting an extract of rose petals with lactic acid bacteria.

Rose is a general term for plants in the genus Rosa of the Rosaceae family. In addition to native varieties, there are many horticultural varieties that have been created by crossbreeding. Horticultural varieties are broadly divided into old roses and modern roses. Old roses include Rosa Centifolia, Rosa Damask, Rosa Gallica, and Rosa Alba, while modern roses include floribunda roses and hybrid tea roses. However, they are not limited to classification by scientific name and species, but are further classified by the appearance and ecological characteristics of the plant body, and it is said that there are as many as 200,000 varieties of roses, including horticultural varieties. In addition, the composition of ingredients contained in the plant body of each variety of rose is diverse, and it is known that the expected biochemical effects differ depending on the variety. Non-Patent Document 1 describes that the content of anthocyanins, flavonols, and carotenoids in rose petals varies depending on the variety, and Patent Document 1 describes that the degree to which rose extract promotes fat production in adipocytes varies depending on the variety. Therefore, in order to obtain a specific effect from the rose used, it is necessary to provide a rose variety that can be strictly distinguished from other varieties by being variety registered or trademarked, and that is stably produced.

As described above, among roses, which are expected to have various biochemical effects depending on the variety, some have been reported to have useful effects on the skin. Patent Document 2 describes that an extract of European rose (Rosa Centifolia) is effective as a skin whitening topical agent. Patent Document 3 describes that a hybrid tea rose or an extract thereof is effective as an elastase activity inhibitor, an antioxidant, and a collagenase activity inhibitor. Although many other rose extracts are known to have useful effects on the skin, none have been known to have a nitrated protein decomposition effect. On the other hand, Patent Document 4 describes that the anti-allergic effect and whitening effect are enhanced by fermenting rose or its extract with lactic acid bacteria. However, there are no known rose extracts that have a nitrated protein decomposition effect, and there have been no known examples of such an effect being enhanced by fermentation with lactic acid bacteria, let alone an example of such an effect being obtained.

Nitrated proteins are a general term for nitrated derivatives that have undergone a post-translational modification called nitration, in which a nitro group is added to the benzene ring in the aromatic amino acid residues of tyrosine and tryptophan that constitute proteins by reactive nitrogen species generated in the body. The tryptophan content in many proteins present in the body is much lower than that of tyrosine, and it is believed that the nitration reaction of proteins occurs mainly in tyrosine residues (Non-Patent Document 2). Non-Patent Document 3 describes that protein nitration affects cell function by causing a decrease in the function of enzymes and tyrosine kinase receptors. Non-Patent Document 4 describes that the accumulation of nitrotyrosine in proteins is involved in a number of diseases, such as arteriosclerosis and ischemic cerebral disease. In addition to these, protein nitration has been reported to be involved in various diseases and symptoms in various tissues in the body, and in the skin, it is known to be involved in yellowing and dullness. Patent Document 5 describes that the cause of yellowing of the skin is not only glycated or carbonylated proteins present in the dermis, as was previously thought, but also nitrated proteins formed by the nitration of amino acids that make up proteins present in the stratum corneum, and that in order to improve various symptoms caused by protein nitration, it is effective to break down the nitrated proteins formed by nitration. Therefore, if nitrated proteins can be broken down, it is expected that various symptoms caused by protein nitration, such as yellowing, will improve.

JP 2019-135213 A JP 2002-029959 A JP 2011-236147 A JP 2010-270152 A JP 2017-181423 A

Front Plant Sci. ,2019,Feb 12;10:123 Front Chem. , 2016, Jan 7; 3:70 Diabetes, 2008, April; 57 (4): 889-98 Science, 2000, Nov 3;290(5493):985-9

The objective of the present invention is to provide a nitrated protein decomposition agent and a skin topical agent that have the effect of improving various diseases and symptoms in various tissues in the body, particularly symptoms caused by protein nitration, such as yellowing of the skin.

The inventors conducted extensive research to solve the above problem, and as a result, solved the above problem by using a fermented product obtained by fermenting an extract of Keihakitami petals with lactic acid bacteria.

The fermented product obtained by fermenting an extract of petals of Rosea keihakitami with lactic acid bacteria has a nitrated protein decomposition effect that was not previously known to be possessed by rose petals or their extracts, or fermented products obtained by fermenting the extracts, and because this effect is extremely high, it is highly effective in improving various symptoms caused by protein nitration, such as yellowing of the skin. In addition, the nitrated protein decomposition agent and topical skin preparation of the present invention can be used continuously or successively to repeatedly decompose nitrated proteins, thereby preventing the accumulation of nitrated proteins, and as a result, suppressing the increase in the nitration rate of proteins in various tissues in the body, including the skin, and thereby preventing various symptoms.

The present invention is explained in detail below.

The Keihakitami used in the present invention is a modern rose of the genus Rosaceae, classified as a hybrid tea rose, and is a horticultural variety also known by the alias "Moonlight", and is registered with the Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 10597 (Keisei Rose Engei, 1999). The fermented product of the present invention is a fermented product (hereinafter referred to as Keihakitami fermented product) obtained by fermenting an extract of Keihakitami petals (hereinafter referred to as Keihakitami extract) with lactic acid bacteria. However, for reasons of industrial production, work, etc., it is acceptable for some parts other than the petals (e.g., calyx, filament, anther, stigma, style, ovary, ovule, pedicel, flower branch) to be included during the preparation process.

The method for preparing the Keihakitami extract to be subjected to fermentation by lactic acid bacteria is not particularly limited, but for example, it can be prepared by extracting the petals of Keihakitami with a solvent. The solvent for obtaining the Keihakitami extract is not particularly limited, but for example, it can be water (purified water, etc.) or an aqueous solution of an inorganic salt (sodium chloride, potassium chloride, magnesium chloride, ammonium carbonate, etc.), a buffer solution (phosphate buffer, acetate buffer, Tris-HCl buffer, carbonate buffer, borate buffer, etc.), an aqueous solution of an inorganic acid (hydrochloric acid, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), an aqueous solution of an organic acid (acetic acid, citric acid, lactic acid, succinic acid, ascorbic acid, fumaric acid, malic acid, etc.), an aqueous solution of a surfactant (saponin, lecithin, etc.), a lower alcohol (methanol, ethanol, propanol, butanol, etc.), or a mixture of these. Examples of solvents that can be used include ketones (acetone, ethyl methyl ketone, etc.), hydrocarbons (propane, butane, hexane, cyclohexane, etc.), ethers (diethyl ether, etc.), glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), halogenated hydrocarbons (dichloromethane, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc.), acetates (ethyl acetate, methyl acetate, etc.), sugars (glucose, fructose, sucrose, maltose, oligosaccharides, etc.), and mixtures of these. The mixing ratio (weight ratio) of petals of Keihakitami to the solvent is generally 1:1 to 1:1000, preferably 1:5 to 1:100, and more preferably 1:10 to 1:50, calculated based on the dry weight of the petals.

The extraction process for obtaining the Keihakitami extract is not particularly limited, but can be carried out, for example, by the following method. First, the petals of Keihakitami to be extracted are immersed or dispersed in the solvent. In this case, the petals may be used raw or may be dried or semi-dried beforehand. The shape is not particularly limited, and the petals may be used as they are after collection, or may be finely chopped or crushed. The extraction process is sufficient to immerse or disperse the Keihakitami petals in the solvent, and there is no need to set a specific extraction time, but a certain extraction time may be appropriately set so that the petals do not rot. Before subjecting the obtained Keihakitami extract to the fermentation process, it is preferable to separate the liquid phase by solid-liquid separation means such as filtration or centrifugation, and remove the immersed or dispersed petals, but this solid-liquid separation process may be omitted if it does not interfere with the fermentation process. After preparation, the Keihakitami extract may be used for fermentation as is, or may be subjected to solvent replacement or purification treatment according to conventional methods, or may be diluted or concentrated to an appropriate concentration if necessary. In some cases, the liquid phase after solid-liquid separation may be solidified according to conventional methods such as spray drying or freeze drying, and may be further pulverized to powder form if necessary before use.

The Keihakitami extract prepared by the above method is preferably sterilized as necessary before being subjected to the fermentation process to remove unwanted bacteria that may hinder fermentation. In this case, the method of removing unwanted bacteria may be a method of washing and sterilizing the petals to be used in advance with sterilizing ethanol or the like, and then extracting with a sterile solvent, or a method of extracting the petals with a solvent, obtaining an extract, and then sterilizing with heat. As the heat sterilization method, an autoclave sterilization method in which heating is performed at 105 to 121°C for 10 to 20 minutes, or an intermittent sterilization method in which the mixture is kept at 80 to 90°C for 60 to 120 minutes and repeated once a day for 2 to 3 days, are generally used. However, this sterilization step is not essential. If it does not hinder the next fermentation step, this sterilization step may be omitted.

The lactic acid bacteria used for fermentation in the present invention is not particularly limited, but from the viewpoint of versatility, it is preferable to use Lactobacillus plantarum. It is preferable to pre-culture in a basic medium before subjecting it to the fermentation process. The culture at this stage can be performed in the usual manner as long as the lactic acid bacteria can grow. The composition of the basic medium for lactic acid bacteria is sufficient if it contains a minimum carbon source, nitrogen source, and phosphorus source, but it is preferable to use a medium developed as a medium that shows good growth of lactic acid bacteria as a whole, such as MRS medium (MRS medium composition: peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween ₈₀ 1g, _K2HPO4 2g, sodium acetate 5g, diammonium citrate 2g, _{MgSO4.7H2O 0.2g} , _{MnSO4.nH2O 0.05g} , purified water 1L). Of course, medium compositions other than those described above can also be applied to the present invention as long as they are substances that lactic acid bacteria can assimilate and grow in. Furthermore, the basal medium may contain various additives, such as aqueous solutions of inorganic salts (sodium chloride, potassium chloride, magnesium chloride, ammonium carbonate, etc.) or buffer solutions (phosphate buffer, acetate buffer, Tris-HCl buffer, carbonate buffer, borate buffer, etc.), aqueous solutions of inorganic acids (hydrochloric acid, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), aqueous solutions of organic acids (acetic acid, citric acid, lactic acid, succinic acid, ascorbic acid, fumaric acid, malic acid, etc.), aqueous solutions of surfactants (saponin, lecithin, etc.), lower alcohols (methanol, ethanol, propanol, butanol, etc.), ketones (acetone, ethyl methyl ketone, etc.), charcoal, etc., and the like. Hydrogen compounds (propane, butane, hexane, cyclohexane, etc.), ethers (diethyl ether, etc.), glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), halogenated hydrocarbons (dichloromethane, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc.), acetates (ethyl acetate, methyl acetate, etc.), sugars (glucose, fructose, sucrose, maltose, oligosaccharides, etc.) or mixtures of these can be added, but when added, it is preferable to add them in a range that does not affect the growth of lactic acid bacteria. Furthermore, in the subsequent fermentation step, the above various components may be contained in the solvent at concentrations that do not affect the fermentation of lactic acid bacteria.

The fermentation process for obtaining the fermented product of Keihakitami is not particularly limited, but for example, the Keihakitami extract prepared by the above method can be inoculated with lactic acid bacteria and cultured for fermentation. The amount of lactic acid bacteria to be inoculated is not particularly limited, but is preferably in the range of 10 ⁵ to 10 ¹⁰ cells/mL of the extract. The fermentation temperature is not particularly limited, but is generally in the range of 5 to 50°C, preferably in the range of 25 to 40°C, which is the optimal temperature for the growth of lactic acid bacteria. The number of days for fermentation is not particularly limited, but is, for example, generally 1 to 15 days at the optimal temperature, preferably 3 to 10 days. Although the fermentation culture can be performed by leaving it to stand, shaking culture or aeration culture can also be performed to shorten the fermentation time. After the above fermentation process is completed, in order to stop the fermentation, it is preferable to subject the fermentation culture liquid that has been subjected to the fermentation process under the above conditions to heat sterilization at 80 to 120°C for about 15 to 120 minutes. The fermentation culture solution after sterilization can be used as it is, or the liquid phase can be separated by a solid-liquid separation means, generally and preferably, such as filtration or centrifugation, to give the fermented product.

After preparation, the fermented product of Keihakitami may be used as it is, or may be purified according to a conventional method, or may be diluted or concentrated to an appropriate concentration if necessary. In some cases, the liquid phase after solid-liquid separation may be solidified according to a conventional method such as spray drying or freeze drying, and may be further pulverized to a powder form if necessary before use.
In addition, when the fermented product of Keihakitami is used as each agent of the present invention, the fermented product of Keihakitami may be used as it is, or may be mixed with a general base. The final form may be any form such as liquid, emulsion, gel, solid, powder, granule, etc., and optional ingredients used in skin external preparations may be appropriately mixed as needed within a range that does not impair the effect. Examples of the optional ingredients include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, preservatives, fragrances, various medicinal ingredients, pH adjusters, neutralizers, etc. Examples of the final form include, for example, skin external preparations, basic cosmetics such as milky lotion, cream, lotion, essence, gel, pack, and facial cleanser, makeup cosmetics such as lipstick, foundation, liquid foundation, and makeup pressed powder, cleansing cosmetics such as facial cleanser, body shampoo, and soap, and bath additives, but are not limited to these.
The amount of the fermented product of Keihakitami in each agent may be appropriately adjusted depending on the desired effect, but is optimally in the range of 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, calculated as evaporation residue.

The decomposition of nitrated proteins in this invention includes not only the decomposition and digestion of nitrated proteins and their constituent amino acids, but also the loss of nitro groups through reduction, oxidation, and other chemical reactions.

The following are examples of the effectiveness test of the fermented product of Keihakitami in the present invention. In addition, examples of application of the fermented product of Keihakitami to topical skin preparations are described, but the present invention is not limited to the examples described here.

<Preparation of lactic acid bacteria to be inoculated into the extract>
Lactobacillus plantarum was used as the lactic acid bacteria. One loopful of the lactic acid bacteria strain was added to 30 mL of MRS medium and cultured at 30°C for 3 days to obtain a lactic acid bacteria culture solution. (MRS medium composition: 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 1 g Tween ₈₀ , ₂ g K2HPO4, 5 g sodium acetate, 2 g ammonium dihydrogen citrate, _0.2 g _MgSO4.7H2O , _0.05 g _MnSO4.nH2O , 1 L purified water)

Preparation of extracts and fermentations
100mL of purified water was added to 10g of pre-dried petals of Keihakitami, and the mixture was extracted at room temperature, after which the petals were removed by filtration to obtain Keihakitami extract. 5mL of the lactic acid bacteria culture was added to the obtained extract and thoroughly stirred. The extract inoculated with lactic acid bacteria was subjected to static culture at 30°C for 3 days, and then sterilized at 105°C for 15 minutes. After sterilization, the solid matter was removed by filtration to obtain Keihakitami fermentation product. As a comparative control, a double delight extract and a double delight fermentation product were prepared from dried petals of Double Delight, another rose variety classified as a hybrid tea rose, in the same manner as the Keihakitami extract and fermentation product.

<Measurement of Nitrated Protein Degradation Activity>
Nitrotyrosine (3-nitrotyrosine), a constituent amino acid of nitrated protein, was dissolved in PBS(-) to prepare a nitrotyrosine solution with a final concentration of 0.2 mM. 250 μL of PBS(-) or nitrotyrosine solution was dispensed into a 96-well plate, and each sample was added to each well so that the final concentration of the extract and fermentation product was 100 ppm, and the final concentration of sodium dithionite was 1 mM (174 ppm). After incubation at 37°C for 72 hours, the absorbance at 450 nm was measured using a plate reader. The value calculated by the following formula was used for analysis as the degree of decomposition of nitrated protein.

Table 1 shows the results of the nitration protein decomposition effect of the fermented Keihakitami product.
In Comparative Example 1, which was added with purified water as a negative control, the degree of decomposition of nitrated proteins was 0.0%, whereas in Comparative Example 2, which was added with sodium dithionite, which is used as a nitrated protein reducing agent in various organic synthesis reactions, in an amount far greater than the equivalent amount relative to nitrotyrosine, the degree of decomposition of nitrated proteins was 53.5% under these conditions. On the other hand, Comparative Example 3, which was added with double delight extract, Comparative Example 4, which was added with double delight fermentation product, and Comparative Example 5, which was added with keihakitami extract, all showed results far below those of Comparative Example 2 and did not show any effect that would be considered to be substantially useful as a nitrated protein decomposition agent. However, surprisingly, only in Example 1, which was added with keihakitami fermentation product, a nitrated protein decomposition effect was found to be greater than that of Comparative Example 2 at a lower concentration than Comparative Example 2.

Next, a formulation example of a skin external preparation containing the Keihakitami fermented product of the present invention is shown, but the present invention is not limited thereto. In the following, the numerical value written after the component name indicates the amount of the ingredient, and each example is expressed as a total of 100% by mass. All parts mean parts by mass, and all % means % by mass. The Keihakitami fermented product shown in each of the following examples is a fermented product obtained by fermenting the extracts extracted with various solvents described below with lactic acid bacteria in the extraction process according to a method similar to [0021], and the amount of the ingredient is shown as a mass % converted to the evaporation residue. However, the extracts extracted with 100% EtOH or 50% EtOH aqueous solution were solvent-substituted with purified water before being subjected to fermentation. The effects of the present application were also confirmed in each example.

(Example 2) Cosmetic cream (mass %)
a) Beeswax: 2.0
b) Stearyl alcohol...5.0
c) Stearic acid...8.0
d) Squalane...10.0
e) Self-emulsifying glyceryl monostearate...3.0
f) Polyoxyethylene cetyl ether (20 E.O.)...1.0
g) Fermented Keihakitami (extraction solvent: purified water) ... 0.001
h) 1,3-butylene glycol...5.0
i) Potassium hydroxide...0.3
j) Preservatives and antioxidants...appropriate amount k) Purified water...remainder Manufacturing method Heat and dissolve a) through f) and keep at 80°C. Heat and dissolve h) through k), keep at 80°C, add to a) through f) and emulsify, then cool to 40°C with stirring. Then add g) and stir to dissolve evenly.

(Example 3) Lotion (% by mass)
a) Fermented Keihakitami (extraction solvent: purified water) ... 1.0
b) Glycerin...5.0
c) Polyoxyethylene sorbitan monolaurate (20 E.O.) ... 1.0
d) Ethanol...6.0
e) Fragrance...appropriate amount f) Preservatives and antioxidants...appropriate amount g) Purified water...remainder Manufacturing method Mix a) through g) and dissolve uniformly.

(Example 4) Emulsion (mass%)
a) Beeswax: 0.5
b) Vaseline...2.0
c) Squalane...8.0
d) Sorbitan sesquioleate...0.8
e) Polyoxyethylene oleyl ether (20E.O.) ... 1.2
f) Keihakitami fermentation product (extraction solvent: 1% citric acid aqueous solution) ... 0.05
g) 1,3-butylene glycol...7.0
h) Carboxyvinyl polymer...0.2
i) Potassium hydroxide...0.1
j) Purified water...the remainder k) Preservatives and antioxidants...appropriate amount l) Ethanol...7.0
Manufacturing method: Heat and dissolve a) through e) and maintain at 80°C. Heat and dissolve g) through k), maintain at 80°C, add to a) through e) and emulsify, then cool to 50°C with stirring. Add f) and l) at 50°C, and stir and cool to 40°C.

(Example 5) Facial cleanser (mass %)
a) Stearic acid...12.0
b) Myristic acid...14.0
c) Lauric acid...5.0
d) Jojoba oil...3.0
e) Glycerin...10.0
f) Sorbitol...15.0
g) 1,3-butylene glycol...10.0
h) POE (20) glycerol monostearate ester...2.0
i) Potassium hydroxide...5.0
j) Water...the remainder k) Chelating agent...appropriate amount l) Fragrance...appropriate amount m) Fermented Keihakitami (extraction solvent: 0.2% citric acid aqueous solution)...0.1
Manufacturing method: Heat and dissolve a) to h) and keep at 70°C. Dissolve i) in j) and add to a) to h) for saponification. Then add k) and l) and cool with stirring. Add m) at 50°C and stir and cool to 40°C.

(Example 6) Essence (mass%)
a) Keihakitami fermentation product (extraction solvent: 100% EtOH) ... 0.01
b) Carboxyvinyl polymer...0.05
c) L-arginine... appropriate amount d) Glycerin... 5.0
e) Polyoxyethylene sorbitan monolaurate (20 E.O.)...1.0
f) Ethanol...6.0
g) Fragrance... appropriate amount h) Preservatives, antioxidants... appropriate amount i) Purified water... remainder Manufacturing method Disperse b) in a part of i), then add c) to adjust the pH to 6.5. Then mix a) to i) and dissolve uniformly.

(Example 7) Cosmetic gel (mass%)
a) Keihakitami fermentation product (extraction solvent: 50% EtOH aqueous solution) ... 0.1
b) Carboxyvinyl polymer...0.5
c) Sodium hydroxide...0.05
d) Methyl paraoxybenzoate...0.1
e) Cyclohexane-1,4-dicarboxylic acid bisethoxydiglycol ... 0.5
f) (eicosanedioic acid/tetradecanedioic acid) polyglyceryl-10...0.5
g) PEG/PPG/Polybutylene Glycol-8/5/3 Glycerin...0.5
h) Polyoxyethylene (60 E.O.) hydrogenated castor oil...0.1
i) Fragrance... appropriate amount j) Preservatives and antioxidants... appropriate amount k) Purified water... remainder Manufacturing method Disperse b) in a part of k), then add c). Then mix a) to k) and dissolve uniformly.

(Example 8) Lipstick (mass %)
a) Fermented Keihakitami (extraction solvent: 0.5% BG aqueous solution) ... 0.01
b) Candelilla wax...9.0
c) Solid paraffin...8.0
d) Beeswax: 5.0
e) Carnauba wax...5.0
f) Lanilin...11.0
g) Castor oil...balance h) Cetyl 2-ethylhexanoate...0.5
i) Isopropyl myristate ester...10.0
j) Titanium dioxide...5.0
k) Red No. 201 ... 0.6
l) Red No. 202...1.0
m) Red No. 223...0.2
n) Fragrance... appropriate amount o) Preservatives, antioxidants... appropriate amount Manufacturing method Disperse j) to m) into a part of g) and process with a roller. Then mix a) to o), heat and melt, then disperse uniformly. After dispersion, pour into a mold, rapidly cool, and make into a stick shape.

(Example 9) Powder foundation (mass %)
a) Fermented Keihakitami (extraction solvent: 5% BG aqueous solution) ... 0.05
b) Squalane...10.0
c) Sorbitan sesquioleate...3.5
d) Preservatives and antioxidants...appropriate amount e) Titanium oxide...13.0
f) Sericite...25.0
g) Talc...balance h) Red iron oxide...0.8
i) Yellow iron oxide...2.5
j) Black iron oxide...0.1
Mix e) to j) and crush in a crusher. Then add a) to d) that have been mixed and dissolved, stir and mix, crush again, and press into a metal dish.

(Example 10) Bath additive (mass%)
a) Keihakitami fermentation product (extraction solvent: 2% glucose aqueous solution) ... 0.5
b) Cyclohexane-1,4-dicarboxylic acid bisethoxydiglycol ... 0.2
c) (eicosanedioic acid/tetradecanedioic acid) polyglyceryl-10...0.2
d) PEG/PPG/Polybutylene Glycol-8/5/3 Glycerin...0.2
e) Sodium bicarbonate...50.0
f) Sodium sulfate... remainder g) Fragrance... appropriate amount h) Preservatives, antioxidants... appropriate amount Manufacturing method Mix a) to h) uniformly.

As described above, it has been confirmed that the fermented product obtained by fermenting the petal extract of Keihakitami with lactic acid bacteria has a remarkable effect of promoting the decomposition of nitrated proteins. Since it is known that nitrated proteins are the cause of yellowing of the skin, it is expected that various symptoms caused by protein nitration will be improved by applying the skin preparation of the present invention to skin with noticeable yellowing.

Claims (4)

A nitrated protein decomposition agent containing a fermented product obtained by fermenting petal extract of Keihakitami (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 10597) with lactic acid bacteria. A skin topical preparation for improving yellowish dullness of the skin caused by nitration, comprising the fermentation product according to claim 1 . 3. The external skin preparation according to claim 2 , wherein the lactic acid bacterium is Lactobacillus plantarum. Use of the fermentation product according to claim 1 or the topical agent according to claim 2 for decomposing nitrated proteins (excluding use in treating humans) .