JP6557166B2 - Nitrated proteolytic agent - Google Patents

Description

The present invention relates to nitrated proteolysis, and more particularly to a nitrated proteolytic agent, a nitrated tyrosine decomposer, and a yellowing dullness improving agent containing Soseki Tea (registered trademark) extract. Hereinafter, the nitrated protein may be simply referred to as nitrated protein, but it is synonymous.

Protein nitration is a post-translational modification of proteins caused by reactive nitrogen species generated in vivo. Nitro groups are added to the benzene rings in the tyrosine and tryptophan residues of the aromatic amino acids that make up proteins. It is a thing. The nitration reaction occurs when the benzene ring in the amino acid undergoes an electrophilic substitution reaction with a nitronium ion (NO2 +) or a nitrogen dioxide radical formed by active 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 at tyrosine residues (Non-patent Document 2).
It is known that when protein nitration occurs, cell functions are affected by causing functional degradation of enzymes and tyrosine kinase receptors (Non-patent Document 3). In addition, nitrotyrosine in proteins is known to accumulate in many diseases associated with aging (such as arteriosclerosis and cerebral ischemic disease), and has been reported to be involved in these diseases (non-patented) Reference 4).

Skin changes with wrinkles, sagging, spots, dullness, etc. with aging.
There are various types of dullness, such as dullness due to reduced transparency (such as due to shadows due to irregularities on the skin surface, due to reduced light transmission due to thickening of the stratum corneum, due to reduced gloss due to irregular reflection on the skin surface), There are dullness due to a decrease in redness of the skin (due to poor circulation), dullness due to uneven skin color (non-uniformity of melanin), dullness due to an increase in yellowness of the skin with aging (Non-patent Document 5).
In particular, the increase in yellowness (b *) of skin color with aging is known as yellowing and is a characteristic skin tone change with aging. Conventionally, the main cause of yellowing was thought to be a glycated product of proteins such as elastin and collagen (Patent Document 1) and a carbonylated protein (Patent Document 2) present in the dermis.

In order to improve various diseases associated with the production of nitrated proteins, a proposal of an effective component for eliminating peroxynitrite (Patent Document 3) and an effective component for supplementing peroxynitrite with a substance having a thiol group (Patent Document 4) ) Has been made. However, all of these only inhibit the production of nitrated proteins, and no improvement is made for the nitrated proteins that have been produced. Therefore, the fundamental solution of various diseases associated with the production of nitrated proteins has not been achieved.

On the other hand, meteorite tea, after harvesting the leaves of Camellia sinensis, is steamed to stop spontaneous fermentation, and then fermented by aerobic mold while sleeping in it, and by anaerobic bacteria while immersed in straw A two-stage fermented tea. As the ingredients contained in Goseki tea, ascorbic acid and catechin compounds are less than other teas, and it has been pointed out that it may contain unique active ingredients. Non-patent document 6), the effect of suppressing the increase in dynamic blood pressure (patent document 5) is known, but the degradation effect of nitrated protein was not known at all.

Chem Res Toxicol. 2009 May; 22 (5): 894-898 Front Chem. 2016 Jan 7; 3: 70. Diabetes. 2008 Apr; 57 (4): 889-98 Science. 2000 Nov 3; 290 (5493): 985-9. Japan Cosmetic Industry Association, Cosmetic Engineering Technical Materials, 101-148 (1995) Mitsuhiko Miyamura, Hironori Moriyama, Shuzo Hamada, Satoko Yokota, Saburo Yoshioka, Daisuke Takuma, Atsuhide Hamada, Yutaka Nishioka. Hyperlipidemia and arteriosclerosis-inhibiting effect of post-fermented tea "Koshiishicha" in cholesterol-loaded rabbits. YAKUGAKU ZASSHI l28: 1037-1044, 2008.

JP 2013-133303 A JP 2012-32287 A JP 2002-326922 A JP 2005-170849 A JP 2014-43406 A

  Under these circumstances, in order to fundamentally solve various diseases associated with the production of nitrated proteins, the inventors do not simply suppress the production but to decompose the nitrated protein that has been produced. However, it was concluded that it would lead to a fundamental solution.

In addition, when research was conducted on the cause of yellowing, the cause of yellowing was not only glycated or carbonylated proteins in the dermis, but also proteins in the stratum corneum. Clarified that the nitrated protein produced by the nitration of the amino acids that make up the protein is a major cause, and further found that the nitrated protein can be decomposed by the meteorite tea extract. It came to.

This invention makes it a subject to provide the component which decomposes | disassembles the nitrated protein which has been completed.

The present inventors have solved the above problems by using a meteorite tea extract.

According to the present invention, the nitrated protein that has been produced can be decomposed, and various symptoms such as arteriosclerosis and cerebral ischemic disease, cellular function decline, and yellowing caused by the nitrated protein can be improved. I can do it.

Relationship between the amount of nitrotyrosine in protein and yellowing Comparison of the degree of degradation of nitrated protein in each test material Comparison of antioxidant effect of each test material

  Before describing the embodiments of the present invention, new knowledge obtained by the present inventors will be described.

<Identification of the cause of yellowing>
The inventors have determined that the cause of yellowing due to the following method is a nitrated protein produced by the nitrated protein in the stratum corneum.

<Experiment method>
Females in their 20s, 30s, 40s and 50s (4-5 people each) who had consented to the measurement content in advance, and the skin color of the cheeks with a spectrocolorimeter (CM-700d, Konica Minolta) Measurement was made and the cheek stratum corneum was collected. The stratum corneum was collected by attaching an adhesive tape (manufactured by 3M) to the cheek and collecting the stratum corneum. The tape to which the stratum corneum is adhered is cut into approximately 3 mm squares, and the elution buffer (100 mM Tris / HCl (pH 8.0), 140 mM NaCl, 0.1% Tween-20, 10 μg / mL aprotinin, 1 The mixture was mixed into a tube containing mM phenylmethylsulfurylfluoride). The mixture was sonicated to elute stratum corneum protein from the tape into the buffer. The elution solution was transferred to a new tube and centrifuged, and the supernatant was collected as a stratum corneum extract sample.
The amount of 3-nitrotyrosine in the stratum corneum extract sample was quantified using NWLSS ^™ Nitrotyrosine ELISA (Northwest Life Science Specialties), and the protein amount of the stratum corneum extract sample was quantified by the BCA method. The amount of 3-nitrotyrosine divided by the amount of protein was defined as the amount of 3-nitrotyrosine per stratum corneum protein.

Formula 1

<Result>
Pearson's correlation test was performed on the relationship between the amount of 3-nitrotyrosine per cheek stratum corneum protein and the yellowness of the skin color (b * value). It was strongly shown that the more nitrated protein in the stratum corneum, the more yellow the skin on the cheeks (FIG. 1).

When this result is analyzed according to age group, the amount of nitrated protein and the yellowness of the skin color (b * value) are higher in the 40-50 generation layer than in the 20-30 generation layer, as shown in Table 1. It was found that the correlation of was high.
From this, it can be said that if the nitrated protein can be decomposed, the yellowing of the skin can be improved, and the effect of the material can be expected to improve the yellowing of the skin in an older age group.

  The meteorite tea used in the present invention is obtained by harvesting the leaves of Camellia sinensis, steaming it to stop natural fermentation, and then rather than aerobic mold while sleeping in it, anaerobic bacteria while soaking A two-stage fermented tea that is fermented. Since it is traditionally produced in Kochi Prefecture, it is possible to use a commercially available product, or it can be produced by the method described in JP-A-2014-43406, JP-A-2009-183272, or the like.

The preparation of the meteorite tea extract is not particularly limited, and for example, it is extracted from a low temperature to a warm temperature using various appropriate organic solvents. 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; and ethyl acetate. One or more of alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and halogenated alkanes such as dichloromethane and chloroform can be used. Among them, water, ethyl alcohol, and a mixed solvent of one or more of 1,3-butylene glycol are particularly suitable.

  The extraction method of the meteorite tea extract that can be used in the present invention is not particularly limited. For example, if it is dried, it is 1 to 1000 times by weight, particularly 10 to 100 times by weight, In this case, it is preferable to carry out at 0 ° C. or higher, particularly 20 ° C. to 40 ° C. for 1 hour or longer, particularly 3 to 7 days. Moreover, you may heat-extract at 60-100 degreeC for 1 hour. Further, the extraction may be performed at a temperature at which the extraction solvent at 10 ° C. or lower does not freeze for 1 hour or longer, particularly 1 to 7 days.

The meteorite tea extract obtained under the above conditions may be used as it is, but if necessary, use it after properly filtering and concentrating and pulverizing. I can do it.

  The blending amount of the meteorite extract in each agent of the present invention is preferably 0.00001 to 20.0% by weight, particularly in the range of 0.001 to 10.0% by weight, in terms of the evaporation dry matter.

  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.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples. Unless otherwise specified, the blending amount is expressed in mass%. Unless otherwise specified, reagent special grade (99.5%) was used for ethanol.

<Preparation of test substance>
A 10-fold weight 50V / V% aqueous ethanol solution was added to the dried plant material, and the mixture was extracted by heating at 60 ° C. for 4 hours. A test substance was prepared by diluting ethanol and water with a weight ratio of 1: 2.5: 96.5 to the dried residue of the extract. The tea leaves used were non-fermented tea (green tea, bancha, hojicha), semi-fermented tea (oolon tea) obtained by semi-fermenting tea leaves with oxidase, fermented tea (tea), tea leaves obtained by completely fermenting tea leaves with oxidase. It is a post-fermented tea (pu-erh tea, meteorite tea) fermented with oxidase and microorganisms.

<Nitrated proteolytic activity test>
3-Nitrotyrosine, a constituent amino acid of nitrated protein, was dissolved in PBS to prepare a solution having a concentration of 0.005% (w / v). PBS or 3-nitrotyrosine solution was dispensed at 250 μL into 96 well-plates, and the test substance and the 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.

Formula 2

Fig. 2 shows the composition of nitrated proteins in the control substance (control) and non-fermented tea extract (green tea, bancha, hojicha), semi-fermented tea (oolong tea), fermented tea (black tea), and post-fermented tea (pu-erh tea). The degree of degradation of 3-nitrotyrosine, an amino acid, remained at about 0-7%.
On the other hand, the meteorite tea extract, which is a post-fermented tea, had a degradation rate of 3-nitrotyrosine, which is a constituent amino acid of nitrated protein, exceeding 20%. From the above, it was shown that the meteorite tea extract has an effect of decomposing nitrated protein.

<Antioxidant effect test>
DPPH Test <Preparation of DPPH Solution> The DPPH solution is prepared by mixing the following components in 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 adjusted to pH 6.1 with 1N NaOH.
(B) 15.7 mg of DPPH (1,1-diphenyl-2-picrylhydrazyl) is dissolved 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 solution.
<DPPH test> 10 μL of test substance or Trolox solution (0.078 mM, 0.156 mM, 0.313 mM, 0.625 mM, 1.25 mM) was added into each well of a 96 well plate, Quickly add 190 μL of DPPH solution and mix. After 10 minutes, the absorbance of each well is measured at 540 nm. A calibration curve of the Trolox concentration and the absorbance value of 540 nm was prepared, and the Trolox equivalent of the test substance was calculated from the absorbance of the test substance at 540 nm.

From FIG. 3, the antioxidant power (Trolox equivalent) of each test substance was highest in the green tea extract, followed by the black tea extract, the hoji tea extract, and the meteorite tea extract. In FIG. 2, since 3-tyrosine-degrading activity, which is a nitrated protein-constituting amino acid, was observed only in the meteorite tea extract, it was considered that there was no relationship between the antioxidant power and the nitrated proteolytic power. At the same time, it was suggested that Soseki tea extract has completely different properties from other tea extracts.

Hereinafter, the formulation example of each agent in this invention is shown. In addition, content is mass%. A manufacturing method is a conventional method. In addition, prescription is a representative example and is not limited to this. Moreover, the density | concentration of the meteorite tea extract in a prescription example is a density | concentration as a dry residue.

<Prescription Example 1: Ointment>
Meteorite tea extract (1,3 butylene glycol 50v / v% solvent, 60 ° C, 4 hours extraction) 0.1
Resorcin 0.5
Octyl paradimethylaminobenzoate 4.0
Butylmethoxybenzoylmethane 4.0
Stearyl alcohol 18.0
Owl 20.0
Glycerin monostearate 0.3
Vaseline 33.0
Perfume Appropriate amount Preservative / Antioxidant Appropriate amount Purified water balance Total 100.0

<Prescription Example 2: Tablet>
Soseki tea extract (ethanol 50v / v% aqueous solution, 25 ° C, 3 days extraction) 5.0
Eggshell calcium 10.0
Lactose 20.0
Starch 7.0
Dextrin 8.0
Hardened oil 5.0
Cellulose remaining total 100.0

<Prescription Example 3: Energy Drink>
Meteorite tea extract (water, 60 ° C, extracted for 4 hours) 1.0
Taurine 3.0
Pyridoxine hydrochloride 0.02
Thiamine sulfide 0.01
Riboflavin 0.003
Anhydrous caffeine 0.05
Purified white sugar 5.0
D-sorbitol solution 2.0
Citric anhydride 0.2
Perfume
Purified water balance 100.0

<Formulation example 4: lotion>
Meteorite tea extract (ethanol solvent, 5 ° C, 1 day extraction) 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
Preservative appropriate amount pH adjuster appropriate amount purified water remaining balance 100.0

<Prescription Example 5: Foundation>
Meteorite tea extract (ethanol solvent, 25 ° C, extracted for 1 day) 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 balance Total 10.0

According to the present invention, since the nitrated protein can be decomposed by using the meteorite tea extract, it can be expected to improve various symptoms caused by the production of nitrated protein.

Claims (3)

Nitrated proteolytic agent containing Goishi tea (registered trademark) extract. A nitrated tyrosine degrading agent containing Soseki Tea (registered trademark) extract. Yellow dullness improving agent containing Soseki Tea (registered trademark) extract.