JP2021147335A - Nitrated protein decomposer - Google Patents

Abstract

To provide an excellent nitrated protein decomposer.SOLUTION: A nitrated protein decomposer contains hibiscus extract. The nitrated protein decomposer can improve various symptoms caused by nitrated protein, such as arteriosclerosis, brain ischemia disease, cell dysfunction, yellow darkening of skin.SELECTED DRAWING: None

Description

The present invention relates to nitrated proteolysis, and more particularly to a nitrated proteolyzer containing a hibiscus extract, a nitrotyrosine degrading agent, and a yellowing improving agent.

Protein nitration is one of the post-translational modifications of proteins caused by active nitrogen species generated in vivo, and a nitro group is added to the benzene ring in the residues of tyrosine and tryptophan, which are aromatic amino acids constituting the protein. It is a protein. The nitration reaction occurs when the benzene ring in an amino acid ^{undergoes an electrophilic substitution reaction with nitronium ions (NO 2+} ) and nitrogen dioxide radicals formed by reactive nitrogen species (Non-Patent Document 1). The content of tryptophan in many proteins existing in the living body is much smaller than that of tyrosine, and it is considered that the nitration reaction of proteins mainly occurs in tyrosine residues (Non-Patent Document 2). It is known that when protein nitration occurs, it affects cell function by causing a decrease in the function of enzymes and tyrosine kinase type receptors (Non-Patent Document 3). In addition, nitrotyrosine in proteins is known to accumulate in a number of age-related diseases (arteriosclerosis, cerebral ischemic disease, etc.), and has been reported to be involved in these diseases (non-patented). Document 4).

With aging, changes such as wrinkles, sagging, stains, and dullness occur on the skin. There are various types of dullness, such as dullness due to decreased transparency (due to shadows due to unevenness on the skin surface, due to decreased light transmission due to thickening of the stratum corneum, due to decreased gloss due to diffused reflection on the skin surface, etc.), There are dullness due to a decrease in redness of the skin (due to poor blood circulation), dullness due to uneven skin color (non-uniformity of melanin), and dullness due to an increase in yellowing of the skin with aging (Non-Patent Document 5). Among them, the increase in skin color yellowing (b *) with aging is known as yellowing, and is a characteristic change in skin color tone with aging. Conventionally, it has been considered that the main cause of yellowing is a saccharified product of a protein such as elastin or collagen (Patent Document 1) or a carbonylated product of a protein (Patent Document 2) existing in the dermal skin. The inventor has clarified that not only the protein in the dermal layer but also the nitrated protein produced by the nitration of the amino acids constituting the protein existing in the stratum corneum is a major cause (Patent Documents 3 and 6). ). Since all post-translational modifications of proteins such as glycation, carbonylation, and nitration have different production and degradation mechanisms, it is necessary to deal with each factor in order to effectively improve yellowing.

Among them, for nitrated proteins, in order to improve various diseases associated with the production of nitrated proteins, we have proposed an effective ingredient that eliminates peroxynitrite (Patent Document 4), and peroxy using a substance having a thiol group. Proposals for effective ingredients that supplement nitrous have been made (Patent Document 5). However, all of these exclusively suppress the production of nitrated proteins, and no improvement has been made to the nitrated proteins that have been produced, which is the basis of various diseases associated with the production of nitrated proteins. No solution was reached. Under such circumstances, the inventor of the present application found a nitrated proteolytic effect in Goishicha (registered trademark) extract, and applied this to the skin to improve the yellowing associated with the presence of the nitrated protein. Found (Patent Document 3, Non-Patent Document 6).

On the other hand, hibiscus is a plant belonging to the genus Confederate rose in the Malvaceae family, and this extract is known to have an antioxidant effect, a saccharified production inhibitory effect (Patent Document 6), a carbonylated product production inhibitory effect (Patent Document 7), and the like. However, the effect of degrading the translational modification of these proteins that have already been produced has not been reported, much less the degrading effect of nitrated protein.

Chem Res Toxicol. , 22 (5): 894-898, 2009 Front Chem. 3:70, 2016 Diabetes, 57 (4): 889-98, 2008 Science, 290 (5493): 985-9, 2000 Japan Cosmetic Industry Association Technical Data, 101, 148, 1995 FRAGRANCE JOURNAL, vol. 45 / No. 8, 2017

JP 2013-133303 JP 2012-32287 JP-A-2017-181423 JP-A-2002-326922 Japanese Unexamined Patent Publication No. 2005-170849 JP-A-11-106336 JP-A-2006-160630

In order to fundamentally solve various diseases associated with the production of nitrated protein, the inventor of the present application is effective not only to suppress the production but to decompose the produced nitrated protein. We came to the conclusion that it would lead to a solution. Furthermore, it was found that the nitrated protein could be directly decomposed by the hibiscus extract, and the invention was completed.

An object of the present invention is to provide an excellent component that directly decomposes a nitrated protein that has already been produced.

The present inventor has solved the above problems by using a hibiscus extract.

According to the present invention, the already produced nitrated protein can be directly decomposed, and various symptoms such as arteriosclerosis, cerebral ischemic disease, decreased cell function, and yellowing caused by the nitrated protein can be directly decomposed. Can be improved.

The hibiscus used in the present invention is not particularly limited as long as it is a plant belonging to the genus Confederate rose in the Malvaceae family (scientific name: Hibiscus sabdariffa L.). For example, Rosel (Hibiscus sabdariffa L.), Mukuge (Hibiscus syriacus), Hibiscus mutabills, Scarlet rosemallow (Hibiscus coccineus), Shoeblackplant (Hibiscus). The part to be used is not particularly limited. For example, petals, grains, stems, leaves, roots or whole plants can be used.

Here, a known method can be used as a method for obtaining the extract. The preparation of the extract is not particularly limited, but for example, the plant extract is extracted from the above hibiscus, preferably a dried powder, using various suitable organic solvents from low temperature to heating. Examples of the extraction solvent 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; ethyl acetate and the like. Alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ethers; one or more of alcohol halides such as dichloromethane and chloroform can be used. Among them, one or more mixed solvents of water, ethyl alcohol and 1,3-butylene glycol are particularly suitable.

The extraction method of the hibiscus extract that can be used in the present invention is not particularly limited. Any part of the hibiscus can be extracted as it is or cut in advance into small pieces, dried and pulverized into a powder. For example, in the case of a dried product, a solvent of 1 to 1000 times by weight, particularly 10 to 100 times the amount is used, and in the case of normal temperature extraction, 0 ° C. or higher, particularly 20 ° C. to 40 ° C. for 1 hour or longer, particularly It is preferably carried out for 3 to 7 days. Alternatively, it may be heat-extracted at 60 to 100 ° C. for 1 hour. Further, the extraction may be carried out at a temperature of 10 ° C. or lower so that the extraction solvent does not freeze, for 1 hour or more, particularly for 1 to 7 days.

The hibiscus extract obtained as described above can be obtained from activated carbon or activated clay, a styrene-divinylbenzene synthetic adsorbent (HP-20: manufactured by Mitsubishi Kasei Corp.), or octadecylsilane-treated silica (Chromatolex ODS: Fuji Silysia Chemical), if necessary. It can be purified by (manufacturing) or the like, and concentrated and powdered products can be used as appropriate. As the blending amount, 0.0001% by weight to 100% by weight can be arbitrarily used in terms of dry weight, depending on the purpose. A preferable blending amount is 0.01% by weight to 10% by weight, and a more preferable blending amount is 0.05% by weight to 5% by weight.

Each agent of the present invention can be used in combination with other components as long as the effects of the present invention are not impaired.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples. Unless otherwise specified, the blending amount is shown in% by mass. Unless otherwise specified, ethanol used was a reagent special grade (99.5%).

<Preparation of test substance>
A 10-fold weight of a 30 (v / v)% ethanol aqueous solution was added to the bulk of the dried plant, and the mixture was heat-extracted at 60 ° C. for 8 hours. Ethanol and water were added to the dry residue of the extract in a weight ratio of 1:25:74 to dilute the extract, which was used as the test substance. The plant progenitors used were Goishicha (registered trademark), hibiscus petals, rosemary leaves, lemongrass stems, tormentilla rhizomes, and honeysuckle petals. Only the lysing solvent of the bulk plant was used as the control substance.

<Nitrated proteolytic activity test>
3-Nitrotyrosine, which is a constituent amino acid of the nitrated protein, was dissolved in PBS (Phosphate Buffered Saline) to prepare a solution having a concentration of 50 mg / L. 250 μL of PBS or 3-nitrotyrosine solution was dispensed into 96-well-plate, and the test substance and control substance were added to each well to a final concentration of 100 ppm. After incubating at 37 ° C. for 72 hours, the absorbance at 450 nm was measured with a plate reader.

From Table 1, in the Goishicha extract, which has already been confirmed to have a nitrated proteolytic effect, the decomposition rate of nitrotyrosine, which is a constituent amino acid of the nitrated protein, was about 14%, whereas rosemary and lemon. It was about 0 to 1% for the extract of glass and tormentilla, and about 6% for the extract of watermelon. On the other hand, in the hibiscus extract, the decomposition rate of nitrotyrosine was about 30%. From the above results, it was shown that the hibiscus extract has a particularly excellent effect of directly degrading nitrotyrosine and nitrated protein. Moreover, since the nitrated protein is a cause of yellowing, a hibiscus extract capable of directly decomposing the nitrated protein can be expected to have a more excellent effect of improving yellowing.

<Antioxidant effect test>
It was evaluated by the DPPH test.
<Preparation of DPPH solution> The DPPH solution was prepared by mixing each of the following components at a ratio of (A) :( B) :( C) = 1: 4: 3 volume.
(A) 5.52 g of MES (2- (N-morpholino) ethanesulfonic acid) is dissolved in 100 mL of water, and the pH is adjusted to 6.1 with 1N NaOH.
(B) Dissolve 15.7 mg of DPPH (1,1-diphenyl-2-picrylhydrazil) in 100 mL of ethanol.
(C) Purified water
<Preparation of Trolox solution> 25 mg of Trolox was dissolved in 10 mL of DMSO (dimethyl sulfoxide) to prepare a 10 mM Trolox solution.
<DPPH test> 10 μL of the test substance or Trolox solution is added to each well of 96 well-plate, and then 190 μL of DPPH solution is rapidly added and mixed. After 10 minutes, the absorbance of each well is measured at 540 nm. A calibration curve of the Trolox concentration and the absorbance value at 540 nm was prepared, and the Trolox equivalent of the test substance was calculated from the absorbance at 540 nm of the test substance.

From Table 2, the antioxidant power (Equal amount of Trolox) of each test substance is about 8 mM for Goishicha extract, about 10 mM for Tormentilla extract, about 6 mM for honeysuckle extract, and other hibiscus, rosemary, and lemongrass extracts. Was 1 mM or less. In view of the results in Tables 1 and 2, it was suggested that there is no relationship between the nitrotyrosine degrading action, which is a nitrated protein-constituting amino acid, and the antioxidant power.

<Saccharification decomposition effect test>
I went as follows.
<Preparation of reaction solution> The reaction solution was prepared by mixing each of the following components at a ratio of (A) :( B) :( C) = 5: 1: 4 volume.
(A) Test substance (B) 10 mM 1-Phenyl-1,2-Propanedione (PPD)
(C) 200 mM phosphate buffer (pH 7.4)
<Saccharification decomposition test> After reacting the mixed reaction solution at 37 ° C. for 8 hours, 1/5 weight of the reaction solution was added with a 2M hydrochloric acid aqueous solution to terminate the reaction. The reaction solution was centrifuged at 3,000 × g for 10 minutes, and the amount of benzoic acid in the supernatant was measured by HPLC.
<Analysis conditions>
Analytical column: Chemcobond 5-ODS-W (150 x 6 mm)
Detection wavelength: 254 nm
Mobile phase: 2 mM EDTA.2Na-0.2% acetic acid / acetonitrile (70/30)
Column temperature: 40 ° C
Flow velocity: 1.0 mL / min

From Table 3, the saccharification cross-linking rate of each test substance was highest in rosemary extract, followed by goishicha extract and tolmentilla extract, honeysuckle extract, lemongrass extract, and hibiscus extract. In view of the results in Tables 1 and 3, it was suggested that there is no relationship between the nitrotyrosine decomposition action and the glycation decomposition action.

Hereinafter, prescription examples of each agent in the present invention will be shown. The content is mass%. The manufacturing method is a conventional method. The prescription is a typical example and is not limited to this. The concentration of the hibiscus extract in the formulation example is the concentration as a dry residue.

<Prescription example 1: Ointment>
Hibiscus petal extract (1,3 butylene glycol 50 (v / v)% solvent, 60 ° C., 4 hours extraction) 0.1
Resorcin 0.5
Octyl paradimethylaminobenzoate 4.0
Butylmethoxybenzoylmethane 4.0
Stearyl alcohol 18.0
Japan wax 20.0
Glycerin monostearic acid ester 0.3
Vaseline 33.0
Perfume Appropriate amount Preservative / Antioxidant Appropriate amount Purified water Remaining total 100.0

<Prescription example 2: Tablets>
Hibiscus leaf extract (ethanol 50 (v / v)% aqueous solution solvent, 25 ° C., 3 days extraction) 5.0
Eggshell calcium 10.0
Lactose 20.0
Starch 7.0
Dextrin 8.0
Hydrogenated oil 5.0
Cellulose balance total 100.0

<Prescription example 3: Energy drink>
Hibiscus petal extract (water, 60 ° C, 4 hours extraction) 1.0
Taurine 3.0
Pyridoxine hydrochloride 0.02
Thiamine sulfide 0.01
Riboflavin 0.003
Anhydrous caffeine 0.05
Purified sucrose 5.0
D-sorbitol solution 2.0
Citric acid anhydride 0.2
Appropriate amount of fragrance
Purified water balance total 100.0

<Prescription example 4: Toner>
Hibiscus stalk extract (water solvent, 60 ° C, 3 hours extraction, HP-20 purification) 0.01
Polyoxyethylene sorbitan monolaurate (20EO) 1.5
1,3-butylene glycol 4.5
Glycerin 3.0
Ethanol 2.0
Sodium hyaluronate (1% aqueous solution) 5.0
Trisodium edetate 0.1
Preservatives Appropriate amount pH adjuster Appropriate amount Purified water Total balance 100.0

<Prescription example 5: Foundation>
Hibiscus grain extract (ethanol solvent, 25 ° C, 1 day extraction) 0.05
Talc 5.0
Sericite 8.0
Titanium oxide 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 Preservative Appropriate amount Purified water Remaining total 100.0

According to the present invention, by using the hibiscus extract, the nitrated protein and nitrotyrosine can be decomposed, and therefore various symptoms caused by their production can be expected to be improved.

Claims (3)

A nitrated proteolytic agent containing a hibiscus extract. A nitrotyrosine degrading agent containing a hibiscus extract. A yellowing inhibitor containing a hibiscus extract.