Nitrated protein reducer

Abstract

To provide an excellent nitrotyrosine reducer that has the effect of inhibiting nitrotyrosine production, which inhibits the new generation of nitrated proteins, as well as the effect of decomposing already generated nitrated proteins; and to provide an agent that can prevent and improve aging changes in skin properties such as the abnormal accumulation of elastic fibers and the decrease in skin elasticity.SOLUTION: The above problem was solved by using Syzygium aromaticum extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the reduction of nitrated proteins, and more particularly to a nitrated protein reducer, a nitrotyrosine reducer, an agent for preventing and improving abnormal accumulation of elastic fibers in the skin, and an agent for preventing and improving loss of elasticity, each containing an extract of Clove. [Background technology]

[0002] Protein nitration is a type of post-translational modification of proteins caused by reactive nitrogen species generated in vivo. It is a reaction in which a nitro group is added to the benzene ring in the aromatic amino acid residues of tyrosine and tryptophan that make up proteins. The nitration reaction occurs when the benzene ring in the amino acid is converted to a nitronium ion (NO2 + ) or nitrogen dioxide radicals (Non-Patent Document 1). Because the content of tryptophan in many proteins present in living organisms is much lower than that of tyrosine, nitration of proteins is thought to occur mainly at tyrosine residues, resulting in the formation of nitrotyrosine in proteins (Non-Patent Document 2).

[0003] It is known that protein nitration causes a decrease in the function of enzymes and tyrosine kinase receptors, affecting cellular functions (Non-Patent Document 3). It has also been reported that nitrotyrosine in proteins accumulates in a number of age-related diseases, such as arteriosclerosis and cerebral ischemia, and is involved in these diseases (Non-Patent Document 4).

[0004] As skin ages, it loses elasticity, resulting in changes such as loss of firmness, sagging, and wrinkles. These changes are particularly pronounced in areas susceptible to chronic UV exposure, such as the face. Skin aging in areas exposed to UV rays is called photoaging, and its prevention and improvement is one of the biggest challenges facing researchers of topical skin preparations. Photoaging skin, in particular, experiences abnormal accumulation of elastic fibers (elastin fibers), which contribute to skin flexibility and elasticity. This abnormality is known to lead to loss of skin strength and flexibility, leading to changes in skin properties in photoaged skin. The present inventors have discovered that nitration, particularly caused by UV rays, causes abnormal accumulation of elastic fibers in photoaged areas, contributing to a decrease in skin elasticity (Non-Patent Document 5).

[0005] The conventionally used agents for improving elasticity loss, loss of firmness, sagging, wrinkles, etc. are mostly intended to increase collagen fiber or elastic fiber or inhibit their decomposition.However, especially those targeting elastic fiber are intended to increase elastic fiber to compensate for the loss of elasticity that occurs in photoaging area by increasing elastic fiber or inhibiting its decomposition.Therefore, they cannot inhibit the abnormal deposition of elastic fiber that occurs in photoaging area, and do not bring about a fundamental improvement in the progress of photoaging.

[0006] Furthermore, in order to improve various diseases associated with the production of nitrated proteins, effective ingredients have been proposed that eliminate peroxynitrite, which nitrates tyrosine residues in proteins (Patent Documents 1 and 2), and effective ingredients that sequester peroxynitrite using substances with thiol groups (Patent Document 3). However, all of these only inhibit the production of nitrated proteins, and do nothing to improve nitrated proteins that have already been produced, so they have not fundamentally resolved the various diseases associated with the production of nitrated proteins. Under these circumstances, the present inventors discovered that Goishicha (registered trademark) extract and hibiscus extract have the effect of decomposing nitrated proteins (Patent Documents 4 and 5). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Chem Res Toxicol., 22(5):894-898, 2009 [Non-patent document 2] Front Chem., 3:70, 2016 [Non-patent document 3] Diabetes, 57(4):889-98, 2008 [Non-patent document 4] Science, 290(5493):985-9, 2000 [Non-Patent Document 5] The 31st IFSCC Congress 2020 Yokohama [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-326922 [Patent Document 2] Japanese Patent Application Publication No. 11-172246 [Patent Document 3] Japanese Patent Application Laid-Open No. 2005-170849 [Patent Document 4] Japanese Patent Application Laid-Open No. 2017-178899 [Patent Document 5] Patent Publication No. 2021-147335 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a nitrotyrosine reducer that not only inhibits the production of nitrotyrosine, which suppresses the new production of nitrated proteins, but also has the effect of decomposing nitrated proteins that have already been produced, and to provide an agent that can prevent and improve aging changes in skin properties such as abnormal accumulation of elastic fibers and decreased skin elasticity. [Means for solving the problem]

[0010] As a result of intensive research into solving the above problems, the inventors of the present application discovered that clove extract is highly effective as a component for inhibiting the new production of nitrotyrosine and for decomposing nitrotyrosine that has already been produced, and have completed the present application.

[0011] That is, the present invention includes the following preferred embodiments. [1] The first invention is: A nitrated protein reducer containing clove extract. [2] The second invention is: A nitrotyrosine reducer containing clove extract. [3] The third invention is: An agent containing clove extract that prevents and improves abnormal accumulation of elastic fibers in human skin. [4] The fourth invention is: An agent containing clove extract that prevents and improves loss of human skin elasticity. [5] The fifth invention is: A candidate agent for preventing and improving abnormal accumulation of elastic fibers in human skin and / or decreased elasticity of human skin, containing an extract of clove tree. [6] The sixth invention is: Use of clove extract for preventing and improving abnormal accumulation of elastic fibers and / or loss of elasticity in human skin. [Effects of the Invention]

[0012] According to the present invention, in addition to inhibiting the new production of nitrated proteins, the nitrated proteins can be decomposed to effectively reduce the amount of nitrated proteins. Furthermore, the nitrated protein reducer of the present invention can prevent or improve various symptoms caused by nitrated proteins, such as arteriosclerosis, cerebral ischemic disease, decreased cell function, and skin symptoms such as decreased skin elasticity due to abnormal accumulation of elastic fibers in photoaged skin. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. Note that the scope of the present invention is not limited to the embodiments described herein, and various modifications can be made without departing from the spirit of the present invention.

[0014] The nitrated protein-reducing agent of the present invention not only acts as an inhibitor of new nitrated protein formation but also as a decomposer of already formed nitrated proteins. The decomposition of nitrated proteins involves not only the digestion or cleavage of nitrated protein-constituting amino acids such as nitrotyrosine by enzymes contained in the reducing agent, but also the conversion of nitrated protein-constituting amino acids such as nitrotyrosine into different substances through oxidation-reduction or other chemical reactions by the components contained in the reducing agent.

[0015] The nitrotyrosine reducer of the present invention not only acts as an inhibitor of new nitrotyrosine production, but also as a decomposer of nitrotyrosine that has already been produced. The decomposition of nitrotyrosine involves not only its disappearance through digestion or cleavage by enzymes contained in the reducer, but also its conversion into a different substance through oxidation-reduction or other chemical reactions by components contained in the reducer.

[0016] The preventive and improving agent of the present invention refers to an agent that can prevent and / or improve aging changes in skin properties such as abnormal accumulation of elastic fibers and decreased skin elasticity caused by nitrated proteins by inhibiting the new production of nitrated proteins and reducing nitrated proteins through decomposition.

[0017] The clove used in this invention is a plant of the Myrtaceae family (scientific name: Eugenia caryophyllata), and its dried flower buds are called cloves or cloves and are widely used as a stomachic herbal medicine and spice. The parts to be used include, but are not limited to, the roots, bark, wood, branches, leaves, flowers, flower buds, fruit, seeds, etc., but it is preferable to use parts containing flower buds.

[0018] Here, known methods can be used to obtain the extract. The preparation of the extract is not particularly limited. For example, the extract can be obtained by extracting the clove tree, preferably dried and powdered, with various suitable organic solvents at low to elevated temperatures. Examples of extraction solvents that can be used include water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and halogenated alkanes such as dichloromethane and chloroform. Mixed solvents of one or more of water, ethyl alcohol, and 1,3-butylene glycol are particularly preferred.

[0019] The extraction method for the clove extract that can be used in the present invention is not particularly limited. Any part of clove can be extracted as is, or pre-cut into small pieces, which are dried and then crushed into powder. For example, in the case of dried clove, a 1 to 1000-fold weight amount, particularly 10 to 100-fold weight amount, of solvent is used. When extracting at room temperature, extraction is preferably performed at 0°C or higher, particularly 20°C to 40°C, for 1 hour or more, particularly 3 to 7 days. Alternatively, extraction can be performed by heating at 60 to 100°C for 1 hour. Alternatively, extraction can be performed at a temperature below 10°C, at which the extraction solvent does not freeze, for 1 hour or more, particularly 1 to 7 days.

[0020] The clove extract obtained as described above can be purified as needed using activated carbon or activated clay, a styrene-divinylbenzene synthetic adsorbent (HP-20: manufactured by Mitsubishi Chemical Corporation), octadecylsilane-treated silica (Chromatorex ODS: manufactured by Fuji Silysia Chemical), or the like, and can be used in a concentrated or powdered form as appropriate. Depending on the purpose, the blending amount can be any amount from 0.0001% to 100% by mass, calculated as the dry solid content. A preferred blending amount is 0.01% to 10% by mass, and a more preferred blending amount is 0.05% to 5% by mass.

[0021] Each agent of the present invention can be used in combination with other ingredients within the scope that does not impair the effects of the present invention. [Example]

[0022] Examples of the present invention will be described in detail below, but the present invention is not limited to these examples. Unless otherwise specified, the blending amounts are expressed in mass %. Unless otherwise specified, special reagent grade ethanol (99.5%) was used.

[0023] <Preparation of test substance> A 10-fold weight of 50 (v / v)% ethanol solution was added to the dried plant extract and heated at 60°C for 8 hours for extraction. The dried extract residue was diluted with ethanol and water at a weight ratio of 1:50:49, and the resulting solution was used as the test substance. The plant extracts used were clove flower buds (cloves), willow mint leaves, oborozuki leaves, Awa bancha tea, Goishicha (registered trademark), and coffee seeds. The control substance was the same dissolution solvent used to prepare the test substance.

[0024] <Nitrotyrosine production inhibition test> L-nitrotyrosine (Fujifilm) was dissolved in 1 M phosphate buffer (pH 7.4) to a concentration of 1 mM, and 1 / 100 of the test substance was added. Peroxynitrite (Dojindo) or 0.3 M NaOH (Fujifilm) solution was added to a final concentration of 100 μM, and the solution was incubated at 37°C for 1 day, after which the amount of nitrotyrosine in the solution was measured by HPLC. <Analysis conditions> Analytical column: Chemcobond 5-ODS-W (150 x 6 mm) Detection wavelength: 355 nm Mobile phase: 10 mM phosphate buffer (pH 2.8) - methanol (9:1 (v / v)) Column temperature: 40℃ Flow rate: 1.0mL / min

[0025] The nitrotyrosine production inhibition rate (%) was calculated from the amount of nitrotyrosine in the obtained solution according to the following formula. The results are shown in Table 1. When the nitrotyrosine production inhibition rate was 20% or more, it was determined that the nitrotyrosine production inhibition effect was achieved, and when it was less than 20%, it was determined that the nitrotyrosine production inhibition effect was not achieved. Furthermore, it can be said that the higher the nitrotyrosine production inhibition rate, the higher the nitrotyrosine production inhibition effect. The results are shown in Table 1.

[0026]

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[0027] [Table 1]

[0028] As shown in Table 1, the clove flower bud and willow mint leaf extracts inhibited nitrotyrosine production by 89.8-100%, with clove flower bud extract showing the highest inhibitory effect on nitrotyrosine production. On the other hand, the extracts of Oborozuki leaf and Awa Bancha tea exhibited nitrotyrosine production inhibition rates of 9.6-11.2%, indicating no inhibitory effect on nitrotyrosine production. These results demonstrate that clove flower bud extract has a particularly excellent effect in inhibiting the de novo synthesis of nitrotyrosine. This suggests that clove flower bud extract is excellent at inhibiting the de novo synthesis of nitrated proteins. Furthermore, since nitrated proteins are the cause of aging skin symptoms such as abnormal elastic fiber accumulation and decreased skin elasticity, clove flower bud extract, which inhibits the production of nitrated proteins, is expected to have a more effective preventative effect on abnormal elastic fiber accumulation and decreased skin elasticity.

[0029] <Nitrotyrosine decomposition activity test> 3-Nitrotyrosine (SIGMA), a constituent amino acid of nitrated proteins, was dissolved in 1 M phosphate buffer (pH 7.4) to a concentration of 400 μM, and 1 / 10 the amount of the test substance was added. The solution was incubated at 37°C for 3 days, and the amount of nitrotyrosine in the solution was measured by HPLC. The test substances were those confirmed to have an inhibitory effect on nitrotyrosine production in Table 1, as well as Goishicha (registered trademark) and coffee seeds, which have already been confirmed to have an inhibitory effect on nitrotyrosine production (see Patent Documents 2 and 4). <Analysis conditions> Analytical column: Chemcobond 5-ODS-W (150 x 6 mm) Detection wavelength: 355 nm Mobile phase: 10 mM phosphate buffer (pH 2.8) - methanol (9:1 (v / v)) Column temperature: 40℃ Flow rate: 1.0mL / min

[0030] The nitrotyrosine decomposition rate (%) was calculated from the amount of nitrotyrosine in the obtained solution according to the following formula. The results are shown in Table 2. Note that when the nitrotyrosine decomposition rate was 5% or higher, it was determined that the nitrotyrosine reduction effect was achieved, and when it was less than 5%, it was determined that the nitrotyrosine reduction effect was not achieved. Furthermore, it can be said that the higher the nitrotyrosine decomposition rate, the greater the nitrotyrosine reduction effect.

[0031]

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[0032] [Table 2]

[0033] As shown in Table 2, in addition to the extract of Goishi Tea (registered trademark), which has already been confirmed to have a nitrotyrosine decomposition activity, the decomposition rate of nitrotyrosine, a constituent amino acid of nitrated protein, was approximately 7.9 to 13.9% in the extract of clove flower buds. In particular, the extract of clove flower buds showed a more pronounced nitrotyrosine decomposition activity than the extract of Goishi Tea (registered trademark). On the other hand, in addition to the willow mint leaf extract, which was confirmed to have a nitrotyrosine production inhibitory effect in Table 1, coffee seeds, which are known to have a nitrotyrosine production inhibitory effect, showed a nitrotyrosine decomposition rate of only 0.1-3.6%, and no nitrotyrosine decomposition effect was confirmed. In other words, it is thought that there is no relationship between the mechanism of action of nitrotyrosine decomposition and the mechanism of action of nitrotyrosine production inhibition. These results demonstrate that the clove flower bud extract has a particularly excellent effect in decomposing nitrotyrosine, and therefore it can be said that the clove flower bud extract has an excellent effect in decomposing nitrated proteins.

[0034] From the above, clove flower bud extract is a highly effective nitrated protein reducer, not only inhibiting the new production of nitrated proteins but also decomposing already produced nitrated proteins, thereby effectively reducing nitrated proteins. Furthermore, since nitrated proteins are the cause of aging skin symptoms such as abnormal accumulation of elastic fibers and decreased skin elasticity, clove flower bud extract is expected to prevent or improve various skin symptoms caused by nitrated proteins, such as arteriosclerosis, cerebral ischemia, decreased cell function, and decreased skin elasticity due to abnormal accumulation of elastic fibers in photoaged skin.

[0035] Below are examples of formulations of each agent of the present invention. The contents are in mass %. The manufacturing method is a conventional method. The formulations are representative examples and are not limited to these. The concentration of the clove extract in the formulation examples is the concentration of the dry residue. The effects of the present application have also been confirmed in these.

[0036] <Prescription example 1: Ointment> Clove flower bud extract (1,3-butylene glycol 50 (v / v)% solvent, extracted at 60°C for 4 hours) 0.1 Resorcinol 0.5 Octyl paradimethylaminobenzoate 4.0 Butyl methoxybenzoylmethane 4.0 Stearyl alcohol 18.0 Rowan 20.0 Glycerin monostearate 0.3 Vaseline 33.0 Fragrance (appropriate amount) Preservatives and antioxidants (appropriate amount) Purified water remainder Total 100.0

[0037] <Prescription Example 2: Tablets> Clove leaf extract (50% (v / v) ethanol aqueous solution, extracted at 25°C for 3 days) 5.0 Eggshell calcium 10.0 Lactose 20.0 Starch 7.0 Dextrin 8.0 Hydrogenated oil 5.0 Cellulose Remaining Total 100.0

[0038] <Prescription example 3: Nutritional drink> Clove rhizome extract (water, 60°C, 4 hours extraction) 1.0 Taurine 3.0 Pyridoxine hydrochloride 0.02 Thiamine sulfide 0.01 Riboflavin 0.003 Caffeine anhydrous 0.05 Refined white sugar 5.0 D-Sorbitol Solution 2.0 Citric acid anhydrous 0.2 Fragrance (appropriate amount) Purified water remainder Total 100.0

[0039] <Formulation example 4: Lotion> Clove flower bud extract (water solvent, 60℃, 3 hours extraction, HP-20 refined) 0.01 Polyoxyethylene sorbitan monolaurate (20E.O.) 1.5 1,3-butylene glycol 4.5 Glycerin 3.0 Ethanol 2.0 Sodium hyaluronate (1% aqueous solution) 5.0 edetate trisodium 0.1 Preservatives (appropriate amount) pH adjuster (appropriate amount) Purified water remainder Total 100.0

[0040] <Formulation example 5: Foundation> Clove fruit extract (ethanol solvent, 25°C, extracted for 1 day) 0.05 Talc 5.0 Sericite 8.0 Titanium dioxide 5.0 Color pigment (appropriate amount) Polyglyceryl Monoisostearate 3.0 Polyoxyethylene hydrogenated castor oil 1.5 Isotridecyl isononanoate 10.0 1,3-butylene glycol 5.0 Antioxidant (appropriate amount) Preservatives: Appropriate amount Purified water remainder Total 100.0

Claims

1. A nitrated protein reducer containing clove extract.

2. A nitrotyrosine reducer containing clove extract.

3. An agent for preventing and improving abnormal accumulation of elastic fibers in human skin, containing extract of clove.

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