JP2012031106A - Carbonylation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new pharmaceutical or cosmetological drug comprising a variety of active ingredients having an action of inhibiting protein carbonylation for inhibiting skin discoloration, especially inhibiting yellowing of the skin.SOLUTION: A protein carbonylation inhibitor consists of: one or more active ingredients selected from an olive leaf extract; a hydrolyzed pea protein; and a lemon extract.

Description

  The present invention relates to a protein carbonylation inhibitor comprising one or more active ingredients selected from olive leaf extract, hydrolyzed pea protein and lemon extract.

  The living body is constantly exposed to oxidative stress (oxygen in the air, ultraviolet rays, harmful substances in the air, irritants in food, etc.). Although the living body originally has a mechanism for scavenging active oxygen, it is known that oxidation proceeds in vivo due to generation of active oxygen exceeding the scavenging ability and reduction of scavenging ability. Among these, proteins are important molecules that not only determine the structure of a living body but also control biological functions such as enzymes, and therefore, studies on the oxidation of biological proteins are being actively conducted. In addition, the oxidation of biological proteins is thought to be involved in organ function decline, and it is known that the oxidation protein increases with aging (Tahara S et al., Mech Ageing Dev. 2001 Apr 15; 122 (4): 415-26).

  “Oxidation” of biological proteins encompasses various reactions caused by active oxygen, and is defined to include not only proteins but also various reactions involving sugars, lipids, and nucleic acids. On the other hand, protein oxidation in a narrow sense means a direct oxidation reaction of a protein by active oxygen that does not involve sugars, lipids, nucleic acids and the like.

  A protein modification reaction involving sugar is called “glycation” and means a protein modification reaction involving sugar oxidation. For example, substances having an aldehyde group or a carbonyl group such as glyoxal, methylglyoxal, and glycol aldehyde, which are generated by oxidative degradation of glucose, are known to modify proteins (Glomb MA et al. J Biol Chem. 1995 Apr 28; 270 (17): 10017-26, Thornalley PJ et al. Biochem J. 1999 Nov 15; 344 Pt 1: 109-16). There are a wide variety of protein modification reactions involving sugars in addition to this pathway, and many final products have been identified.

  On the other hand, protein modification reactions involving lipids are called “protein modification reactions related to lipid peroxidation reactions” and the like. When lipids are exposed to active oxygen, highly reactive aldehydes (carbonyl compounds) such as acrolein, 4-hydroxy-2-nonenal, and malondialdehyde are generated. The reaction in which these intermediate products modify proteins is also referred to as “carbonylation”.

  It has been reported that carbonylation of biological proteins is involved in various diseases and symptoms. Carbonylated proteins are known to be detected in various tissues in the body, and for example, it is reported that they generally increase with age in the liver and brain (Stadtman ER et al., EXS. 62: 64-72, 1992, Levine RL et al. Free Radic Biol Med. 2002 May 1; 32 (9): 790-6). In addition, it has been reported that carbonylated protein increases in progeria, etc. that appear with accelerated aging (Stadtman ER et al., J biol Chem. 262: 5488-5491, 1987). Has been studied. The presence of carbonylated protein is also known in the skin, and accumulation in the horny layer of the exposed area and in the dermis of the photoaging site has been reported (Fujita H. et al., Skin Res Tech. 13: 84- 90, 2007; Sander, CS et al., J Invest Dermatol. 118: 618-25, 2002). Specifically, it has been reported that the carbonylated protein in the exposed stratum corneum causes changes in physical properties such as a decrease in optical transmission (Iwai I. et al., Int J Cosmet Sci. 30: 41- 46, 2008), and dermal carbonylated proteins have been reported to accumulate depending on the severity of photoelastic fibrosis (Tanaka, N. et al., Arch Dermatol Res. 293: 363-367). , 2001; Sander, CS et al., J Invest Dermatol. 118: 618-25, 2002).

  Thus, carbonylation of proteins occurs non-enzymatically and non-specifically, causing aging in all tissues. It is thought that it causes wrinkles, sagging, dullness in the skin, causes functional deterioration in the internal organs and the brain, and may cause various diseases. From such a background, development of various pharmaceutical or cosmetic agents having an action of suppressing protein carbonylation is desired.

Tahara S et al., Mech Ageing Dev. 2001 Apr 15; 122 (4): 415-26 Glomb MA et al. J Biol Chem. 1995 Apr 28; 270 (17): 10017-26 Thornalley PJ et al. Biochem J. 1999 Nov 15; 344 Pt 1: 109-16 Stadtman E.R. et al., EXS. 62: 64-72, 1992 Levine RL et al. Free Radic Biol Med. 2002 May 1; 32 (9): 790-6 Stadtman E.R. et al., J biol Chem. 262: 5488-5491, 1987 Fujita H. et al., Skin Res Tech. 13: 84-90, 2007 Sander, C.S. et al., J Invest Dermatol. 118: 618-25, 2002 Iwai I. et al., Int J Cosmet Sci. 30: 41-46, 2008 Tanaka, N. et al., Arch Dermatol Res. 293: 363-367, 2001 Sander, C.S. et al., J Invest Dermatol. 118: 618-25, 2002

  Since carbonylation of biological proteins occurs non-enzymatically and non-specifically, it causes aging in all tissues. It is thought that it causes wrinkles, sagging, dullness in the skin, causes functional deterioration in the internal organs and the brain, and may cause various diseases. Therefore, an object of the present invention is to provide a novel pharmaceutical or cosmetic agent containing various active ingredients having an action of suppressing protein carbonylation.

  The present inventor has now completed the present invention based on the surprising finding that olive leaf extract, hydrolyzed pea protein and lemon extract significantly suppress carbonylation of protein.

This application includes the following inventions:
[1] A protein carbonylation inhibitor comprising one or more active ingredients selected from olive leaf extract, hydrolyzed pea protein, and lemon extract.
[2] The protein carbonylation inhibitor according to [1] for preventing or treating a symptom or disease related to carbonylation of a biological protein.
[3] The protein carbonylation inhibitor according to [2], wherein the symptom or disease related to carbonylation of a biological protein is yellowing of the skin.

  Suppressing carbonylation of biological proteins, thereby making it possible to prevent or treat various symptoms or diseases related to carbonylation of biological proteins.

The influence with respect to the yellowing of the dermis model by a control (phosphate buffer), D-ribose, glyoxal, 4-hydroxy-2-nonenal, and acrolein is shown. The action which suppresses yellowing of the dermis model by pyridoxine hydrochloride is shown. The action which suppresses yellowing of the dermis model by an olive leaf extract is shown.

  In the present specification, “carbonylation” of a protein means a reaction in which a carbonyl compound such as aldehyde generated by lipid peroxidation or the like modifies a protein nonspecifically and nonenzymatically. When lipids are exposed to active oxygen, lipid peroxides are formed and further decomposed to produce highly reactive aldehydes (carbonyl compounds) such as acrolein, 4-hydroxy-2-nonenal, and malondialdehyde. . These intermediate products modify proteins because they are very reactive. The various substances resulting from the modification are called lipid peroxidation end products (ALEs). On the other hand, a protein modification reaction involving the oxidation of sugar is called “glycation” and is a reaction derived from sugar, which is different from a carbonylation reaction. As a reaction route known as “saccharification”, for example, modification of a protein with glyoxal, methylglyoxal, or glycolaldehyde caused by oxidative degradation of glucose is known (Glomb MA et al. J Biol Chem. 1995). Apr 28; 270 (17): 10017-26, Thornalley PJ et al. Biochem J. 1999 Nov 15; 344 Pt 1: 109-16). There are a wide variety of protein modification reactions involving sugars in addition to this pathway, and many final products have been identified. These are called final glycation products (AGEs).

  Although some ALEs have been found to be partly in common with AGEs, they are different from AGEs in that ALEs are reactions not related to “sugar”.

  The state in which a substance having an aldehyde group or a carbonyl group is generated from “sugar” or “lipid” by active oxygen is called carbonyl stress. In a broad sense, “carbonylation” is used in the same meaning as “oxidation”. However, as used herein, “carbonylation” refers to a protein modification reaction involving aldehydes generated by lipid peroxidation and decomposition (reaction generated by ALEs), and is related to sugar. It is distinguished from the reaction ("glycation").

  In addition, all the reactions generated by substances known as AGEs may be referred to as saccharification in a broad sense. Since it was found that a compound similar to the compound that was previously identified as AGEs is also generated from the reaction pathways related to lipids, “glycation” is used in the same meaning as “oxidation” in the broad sense. However, “glycation” as used herein refers to a protein modification reaction involving sugar, and is distinguished from “carbonylation”.

  The present invention is based on the surprising finding that such protein carbonylation is significantly suppressed by olive leaf extract, hydrolyzed pea protein and lemon extract.

Olive leaf extract Olive (scientific name: Olea europaea Linne) belongs to the magnoliaceae genus, and is now distributed in places with relatively high temperatures around the world. is there. Olive oil obtained by squeezing ripe fruits is widely used for edible, medicinal, and cosmetic applications, and olive leaves are known for their antiseptic, antipyretic and antihypertensive effects, and in recent years have been used as raw materials for tea .

  The method for extracting an active ingredient from olive leaves is not particularly limited, but an extraction method using a solvent is preferred. When extracting, you can use the olive leaves as they are, but if you use pulverized and shredded powders for extraction, you can extract active ingredients with high extraction efficiency in a short time under mild conditions. It can be carried out.

  The extraction temperature is not particularly limited, and may be set as appropriate according to the size of the pulverized olive leaf and the type of solvent. Usually, it is set within the range from room temperature to the boiling point of the solvent. Further, the extraction time is not particularly limited, and may be set as appropriate according to the size of the pulverized olive leaf, the type of solvent, the extraction temperature, and the like. Furthermore, at the time of extraction, stirring may be performed, it may stand still without stirring, and an ultrasonic wave may be added.

  For example, the olive leaf extract can be extracted at room temperature or from 80 ° C to 100 ° C by immersing the olive leaf in a solvent. The filtrate obtained by the extraction treatment can be used as a protein saccharification inhibitor as it is or after concentration or drying as necessary. In this extraction process, olive leaves may be chopped or crushed. In addition, raw olive leaves or dried olive leaves may be used, or roasted olive leaves may be used. The roasting method is not particularly limited, and examples thereof include a method of roasting at 80 ° C. to 120 ° C. for 0.5 hour to 2 hours.

  The type of solvent used for extraction is not particularly limited, but water (including hot water), alcohol (for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol) , Glycol (eg 1,3-butylene glycol, propylene glycol), glycerin, ketone (eg acetone, methyl ethyl ketone), ether (eg diethyl ether, dioxane, tetrahydrofuran, propyl ether), acetonitrile, ester (eg ethyl acetate, butyl acetate) An aliphatic hydrocarbon (for example, hexane, heptane, liquid paraffin), an aromatic hydrocarbon (for example, toluene, xylene), a halogenated hydrocarbon (for example, chloroform), or a mixed solvent of two or more of these is preferable.

  By such an extraction operation, the active ingredient is extracted from the olive leaf and dissolved in the solvent. The solvent containing the extract may be used as it is as a protein carbonylation inhibitor, or may be used after standing for several days and aging. Furthermore, you may use for protein carbonylation inhibitor, after adding conventional refinement | purification processes, such as sterilization, washing | cleaning, filtration, decoloring, and deodorizing. Moreover, you may use for a protein carbonylation inhibitor, after concentrating or diluting as needed. Furthermore, all the solvent may be volatilized to form a solid (dried product) and then used as a protein carbonylation inhibitor, or the dried product may be redissolved in an arbitrary solvent and used as a protein carbonylation inhibitor. Also good.

  In addition, it is also possible to employ | adopt the extraction method using a supercritical fluid as an extraction method of an olive leaf extract. The kind of supercritical fluid is not particularly limited, and examples thereof include nitrogen dioxide, ammonia, ethane, propane, ethylene, methanol, ethanol and the like. In addition, since olive leaf extract is marketed from Maruzen Pharmaceutical Co., Ltd., you may use this.

Lemon Extract Lemon (scientific name: Citrus limon Burmann fil (Rutaceae)) is an evergreen shrub belonging to the genus Citrus mandarin. As the lemon extract used in the protein saccharification inhibitor of the present invention, an extract of lemon fruit (fruit and / or peel) is most preferable, but it is also effective for lemon leaves, stems, branches, flowers, bark, roots, etc. Since ingredients are included, any one or more of these can be used.

  For example, lemon fruit contains a large amount of vitamin C, citric acid, and the like, but the method for extracting active ingredients from lemon is not particularly limited. However, an extraction method using a solvent is preferred. When extracting, lemon can be used as it is, but the active ingredient is extracted with high extraction efficiency in a short time under mild conditions when it is pulverized into granules or powder. be able to.

  The extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product of lemon, the type of solvent, and the like. Usually, it is set within the range from room temperature to the boiling point of the solvent. Further, the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product of lemon, the type of solvent, the extraction temperature, and the like. Furthermore, at the time of extraction, stirring may be performed, it may stand still without stirring, and an ultrasonic wave may be added.

  The type of solvent used for extraction is not particularly limited, but water (including hot water), alcohol (for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol) , Glycol (eg 1,3-butylene glycol, propylene glycol), glycerin, ketone (eg acetone, methyl ethyl ketone), ether (eg diethyl ether, dioxane, tetrahydrofuran, propyl ether), acetonitrile, ester (eg ethyl acetate, butyl acetate) An aliphatic hydrocarbon (for example, hexane, heptane, liquid paraffin), an aromatic hydrocarbon (for example, toluene, xylene), a halogenated hydrocarbon (for example, chloroform), or a mixed solvent of two or more of these is preferable.

  By such an extraction operation, the active ingredient is extracted from the lemon and dissolved in the solvent. The solvent containing the extract may be used as it is as a protein carbonylation inhibitor, or it may be used as a protein carbonylation inhibitor after adding conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. Good. Moreover, you may use for a protein carbonylation inhibitor, after concentrating or diluting as needed. Furthermore, all the solvent may be volatilized to form a solid (dried product) and then used as a protein carbonylation inhibitor, or the dried product may be redissolved in an arbitrary solvent and used as a protein carbonylation inhibitor. Also good.

  In addition, since the extract of lemon is marketed from Koei Kogyo Co., Ltd. (brand name: peach fruit lemon), this can also be used. Moreover, since the active ingredient similar to an extract is contained also in the pressing liquid obtained by pressing lemon, the pressing liquid of lemon can also be used instead of an extract.

  Here, a method for extracting an extract from lemon will be described with an example. When extracting an extract from the fruit of a lemon, although the method similar to the method of extracting an extract from the olive leaf mentioned above can also be employ | adopted, it is preferable to employ | adopt a pressing method. When extracting an extract from parts other than fruits, such as a leaf and a stem, it is preferable to employ a method similar to the above-described method of extracting an extract from olive leaves. In the extraction process from leaves, stems, etc., the lemon may be chopped or crushed. Moreover, the raw or dried thing may be used and the roasted thing may be used.

Hydrolyzed pea protein Hydrolyzed pea protein is a polypeptide obtained by hydrolyzing pea protein. It contains highly acidic amino acids (aspartic acid, glutamic acid) and basic amino acids (arginine, histidine, lysine). Including many. Peas (scientific name: Pisum sativum L.) are legumes that are widely cultivated for food and can be hydrolyzed to obtain various hydrolyzed solutions. Promois WJ (trade name) is a hydrolyzed solution of pea protein having an average molecular weight of about 500, commercially available from Seiwa Kasei Co., Ltd., and can also be used as the hydrolyzed pea protein.

  In the protein carbonylation inhibitor of the present invention, the blending ratio of the olive leaf extract, lemon extract, and hydrolyzed pea protein is not particularly limited and can be arbitrarily set. However, in order to make the carbonylation inhibitory ability of the protein carbonylation inhibitor of the present invention more excellent, the blending ratio of the extract is 0.00001% by mass or more and 10% by mass with respect to the whole protein carbonylation inhibitor. % Or less, more preferably 0.001% by mass or more and 5% by mass or less, and further preferably 0.01% by mass or more and 1% by mass or less.

  The dose, usage, and dosage form of the protein carbonylation inhibitor of the present invention can be appropriately determined according to the purpose of use. For example, the administration form of the protein carbonylation inhibitor of the present invention may be oral, parenteral, external use and the like. Examples of the dosage form include oral preparations such as tablets, powders, capsules, granules, extracts, and syrups, or parenteral preparations such as injections, drops, and suppositories, ointments, creams, emulsions, and lotions. And external preparations such as packs and bath preparations.

  Further, in the protein carbonylation inhibitor of the present invention, in addition to the active ingredient, for example, cosmetically or pharmaceutically acceptable excipients, moisture-proofing agents, preservatives, reinforcing agents, thickeners, emulsifiers, Antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily ingredients, UV absorbers, surfactants, thickeners, alcohols, powders commonly used in cosmetics, etc. Components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

  Furthermore, when the protein carbonylation inhibitor of the present invention is used as an external preparation for skin, auxiliary agents commonly used for external preparations for skin, such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate Sequestering agents such as gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or their derivatives Drugs such as salts, whitening agents such as vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, sugars such as glucose, fructose, mannose, sucrose, trehalose, retinoic acid, retinol, retinol acetate, palmiticin Vitamin A such as retinol may also be appropriately blended.

  It is known that carbonylation of biological proteins is related to various symptoms or diseases. Examples of these symptoms or diseases include aging, visceral function and brain function deterioration, progeria, and photoelastic fibrosis. Psoriasis, atopic dermatitis, wrinkles, spots, sagging skin, and the like. Therefore, the carbonylation inhibitor of the present invention is extremely effective for preventing or treating these symptoms or diseases related to carbonylation of biological proteins.

  Furthermore, the present inventors have obtained a surprising finding that carbonylation of biological proteins, particularly proteins present in the dermis, is closely associated with skin discoloration, particularly skin yellowing. Skin color is one of the important cosmetic elements, and it is known that it is determined by the optical properties of tissues constituting the skin and various pigment components in the skin tissues such as melanin and hemoglobin. Yellowing of the skin is also known as yellowing that causes the skin to lose its transparency, causing a cosmetic problem. However, the relationship between carbonylation of biological proteins and skin discoloration, particularly skin yellowing, has not been known so far.

  Therefore, the protein carbonylation inhibitor of the present invention is extremely useful for suppressing skin discoloration, particularly skin yellowing.

  The invention is illustrated in more detail by the following examples.

Example 1. For the test for evaluating the carbonylation-inhibiting effect of the extract, a 96-well plate previously coated with type I collagen was used (Corning Co., Ltd., code number: NCO3585). As a reagent for inducing carbonylation, acrolein (AccuStandard), which is an aldehyde derived from lipid peroxide, was used. When acrolein is allowed to act on a protein, a carbonyl group is introduced. The introduced carbonyl group was detected by using a hydrazino group (—NHNH ₂ ) and biotin-hydrazide (PIERCE code No. 21339) bound to the carbonyl group. The carbonylation-inhibiting effect of each extract of lemon juice extract, olive leaf extract and hydrolyzed pea protein is compared with the carbonyl group detection efficiency in the presence and absence of these extracts. We examined whether it can be suppressed.

  Specifically, a sample solution was prepared by dissolving 300 μM acrolein and each extract. The concentration of each extract was set to 0.01% to 0.3%. In the sample solution not containing the extract, 300 μM acrolein and the same amount of solvent as the extract were dissolved (positive control). 100 μL of these sample solutions were dispensed into each well of the collagen plate and reacted at 37 ° C. overnight. In addition, the test was similarly performed about the solution which does not contain acrolein but also contains the same amount of solvent as the extract (negative control).

  The plate reacted overnight was washed with a phosphate buffer (PBS-T) containing a surfactant (0.1% Tween 20). Thereafter, 100 μL of a 0.1 μM biotin-hydrazide solution prepared with 100 mM 2-morpholinoethanesulfonic acid monohydrate (MES) buffer was dispensed into each well and reacted at room temperature for 2 hours. After the reaction, the plate was washed with PBS-T buffer. Thereafter, 100 μL of 0.1 μg / mL horseradish peroxidase (HRP) -labeled avidin (Vector A-2004) was dispensed into each well and reacted at 37 ° C. for 1 hour. Further, after washing with PBS-T buffer, a substrate solution containing the hydrochloride (TMB) in 3,3 ′, 5,5′-tetramethylbenzidine which is a substrate for HRP (Bio-Rad, product number 172) The color was developed at -1066). To stop the color reaction, the reaction was stopped with 1M sulfuric acid solution. Then, the light absorbency in wavelength 450nm was measured, and the quantity of the protein in which the carbonyl group was introduce | transduced was quantified.

The effect of inhibiting carbonylation was expressed by (absorbance of sample−absorbance of negative control) / (absorbance of positive control−absorbance of negative control). Each concentration of each sample was evaluated at n = 5. These average values and standard deviation values were determined, t-tests were performed on two independent groups, P values were determined, and significant differences were tested. Statistical significance was defined as “*” having a significant difference of p <0.05 and “**” having a significant difference of p <0.01.

  From the above results, it can be seen that each extract of lemon juice extract, olive leaf extract and hydrolyzed pea protein significantly suppresses carbonylation of the protein. In addition, the peach fruit lemon (brand name) marketed from Koei Kogyo Co., Ltd. was used for evaluation of a lemon juice extract. For the evaluation of the olive leaf extract, an olive leaf extract commercially available from Maruzen Pharmaceutical Co., Ltd. was used. For the evaluation of hydrolyzed pea protein, Promois WJ (trade name) commercially available from Seiwa Kasei Co., Ltd. was used.

Example 2. Examination of relationship between protein carbonylation and skin yellowing Preparation of dermis model Derbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) isolated from human dermis And cultured. Dermal fibroblasts were collected by trypsin-EDTA treatment. As a final concentration, collagen solution (Koken) was diluted with a medium so as to be a 0.1% type I collagen solution containing 1.0 × 10 ⁵ cells / mL of fibroblasts to neutralize collagen. Gelled. For type I collagen, a solution extracted from bovine dermis with acetic acid was used. After neutralization gelation, the medium was changed once every 2-3 days and cultured for 7 days. Since dermal fibroblasts have the property of attracting surrounding collagen fibers, the collagen gel concentrated during the culture and contracted to about 1/3 of the original size. A sufficiently densified contracted collagen gel was immersed in sterilized water, and the fibroblasts surviving in the gel were killed by a hypotonic treatment. The sterilized water was exchanged 10 times or more, and the medium and soluble components were sufficiently removed to produce a gel formed only of dermal components not containing living cells, which was used as a dermis model.

Examination of yellowing factor Using the dermis model described above, the effects of glycation and carbonylation on dermis protein on the dermis model were compared. For glycation of the protein in the dermis model, D-ribose (Wako Pure Chemical Industries, Ltd.), which is a reducing sugar having a higher reactivity than glucose, and glyoxal (Alfa Aesar) produced by autooxidation of glucose were used. For carbonylation, acrolein (AccuStandard) and 4-hydroxy-2-nonenal (Oxis International), which are aldehydes derived from lipid peroxides, which are known to exist in the skin, were used. The dermis model prepared above is immersed in 2.5 mL of a solution containing 200 mM D-ribose, 10 mM glyoxal, 10 mM acrolein and 10 mM 4HNE prepared with a phosphate buffer, and incubated at 37 ° C. for 5 days. And saccharification and carbonylation were induced experimentally. The color change of the dermis model was measured with Konica Minolta spectrocolorimeter CM-2600d diffuse illumination and 8 ° direction light reception φ3 mm. At the time of measurement, the dermis model was taken out from the solution, washed thoroughly with a phosphate buffer, and then placed on a white calibration tile (CR-A43 (1849-701)) for measurement. CIE L ^* a ^* b ^* color system calculated from the obtained spectral reflection spectrum by b ^* was used as an index of yellowness. The change in yellowness (yellowing) was evaluated using a dermis model immersed in a phosphate buffer as a control.
The obtained results are shown in FIG. As can be seen from FIG. 1, when carbonylation of a protein in the dermis model is induced, the yellowing (yellowing) is more significant than glycation, which has been considered to be one of the major factors of skin yellowing so far. It was shown to induce

Inhibition of yellowing of the dermis model by pyridoxine hydrochloride We investigated whether suppression of carbonylation would inhibit protein yellowing in the dermis model. The dermis model produced above was immersed in a solution (control) containing only 1 mM acrolein prepared with a phosphate buffer and 2.5 mL of a solution containing 1 mM acrolein and a candidate substance. As a candidate substance, 10 mM pyridoxine hydrochloride, which is known to have a carbonylation-inhibiting effect, was used. Thereafter, the dermis model treated as described above was incubated at 37 ° C. for 5 days. The solution was changed to a freshly adjusted solution every day. The color change of the dermis model was measured with Konica Minolta spectrocolorimeter CM-2600d diffuse illumination and 8 ° direction light reception φ3 mm. At the time of measurement, the dermis model was taken out of the solution and placed on a white calibration tile (CR-A43 (1849-701)). B ^* (yellowishness) based on the CIE L ^* a ^* b ^* color system calculated from the obtained spectral reflection spectrum was used as an index of yellowing. The obtained results are shown in FIG.

  As can be seen from FIG. 2, it was shown that the yellowing of the skin was suppressed by suppressing the carbonylation of the dermal protein.

Example 3 Evaluation of Skin Yellowing Inhibition Action by Olive Leaf Extract As an example of evaluation, the yellowing inhibition of olive leaf extract (Maruzen Pharmaceutical Co., Ltd.) was evaluated. Olive (Olea europaea Linne) belongs to the magnoliaceae genus, and is an evergreen tree that is cultivated mainly in Shodoshima in Japan where temperatures are relatively high in various parts of the world. Olive oil obtained by squeezing ripe fruits is widely used for edible, medicinal, and cosmetic applications, and olive leaves are known for their antiseptic, antipyretic and antihypertensive effects, and in recent years have been used as raw materials for tea . However, it has not been known so far that olive leaf extract has a carbonylation-inhibiting action. Since the olive leaf extract is colored itself, the same experiment as in Example 2 was performed by using activated carbon from which the pigment component was removed so as not to hinder the evaluation of the color. Specifically, the dermis model prepared above was immersed in a solution (control) containing only 1 mM acrolein prepared with a phosphate buffer and 2.5 mL of a solution containing 1 mM acrolein and 10% olive leaf extract. . Thereafter, the dermis model treated as described above was incubated at 37 ° C. for 6 days. The solution was changed to a freshly adjusted solution every day. The color change of the dermis model was measured with Konica Minolta spectrocolorimeter CM-2600d diffuse illumination and 8 ° direction light reception φ3 mm. At the time of measurement, the dermis model was taken out of the solution and placed on a white calibration tile (CR-A43 (1849-701)). B ^* (yellowishness) based on the CIE L ^* a ^* b ^* color system calculated from the obtained spectral reflection spectrum was used as an index of yellowing.

As can be seen from FIG. 3, yellowing of the dermis model was suppressed, and a significant suppression of b ^* increase was observed. This indicates that the olive leaf extract is very likely to have an action of suppressing skin yellowing.

Formulation Example A formulation example in which the carbonylation inhibitor of the present application is blended is shown below. In addition, this prescription example is an example to the last, and this invention is not limited to this prescription example.

Formulation Example 1: Vanishing cream (O / W type, soap + nonionic surfactant combination)
Agent: Carbonylation inhibitor (hydrolyzed pea protein) of the present application 0.1% by mass
Oil content: Stearic acid 8.0
Stearyl alcohol 4.0
Butyl stearate 6.0
Moisturizer: Propylene glycol 5.0
Surfactant: glyceryl monostearate 2.0
Alkali: Potassium hydroxide 0.4
Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Purified water: 74.5

Manufacturing method Add water, chemicals, moisturizer, and alkali to purified water to prepare an aqueous phase, then heat and adjust to 70 ° C. After heating and dissolving the oil, add a surfactant, preservative, antioxidant, and fragrance, and adjust to 70 ° C. This is added to the previous aqueous phase and pre-emulsified. After making the emulsified particles uniform with a homomixer, deaeration, filtration and cooling are performed.

Formulation Example 2: Emollient cream (O / W type)
Agent: Carbonylation inhibitor (olive leaf extract) of the present application 0.1% by mass
Oil content: Stearyl alcohol 6.0
Stearic acid 2.0
Hydrogenated lanolin 4.0
Squalane 9.0
Octyldodecanol 10.0
Moisturizer: 1,3 Butylene glycol 6.0
PEG1500 4.0
Surfactant: POE (25) cetyl alcohol ether 3.0
Glycerol monostearate 2.0
Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Purified water: 53.9

Manufacturing method Add a chemical and moisturizing agent to purified water to prepare an aqueous phase, and heat and adjust to 70 ° C. After heating and dissolving the oil, add a surfactant, preservative, antioxidant, and fragrance, and adjust to 70 ° C. This is added to the previous aqueous phase, and the emulsified particles are made uniform with a homomixer, followed by deaeration, filtration and cooling.

Formulation Example 3: Emollient cream (O / W type)
Drug: Carbonylation inhibitor (hydrolyzed pea protein) of the present application 0.1% by mass
Oil content: Cetyl alcohol 5.0
Stearic acid 3.0
Vaseline 5.0
Squalane 10.0
Glycerol triethyl 2-ethylhexanoate 7.0
Ester humectant: Dipropylene glycol 5.0
Glycerin 5.0
Surfactant: Propylene glycol monostearic acid 3.0
ester
POE (25) cetyl alcohol ether 3.0
Alkali: Triethanolamine 1.0
Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Purified water: 52.9

Manufacturing method Add water, chemicals, moisturizer, and alkali to purified water to prepare an aqueous phase, then heat and adjust to 70 ° C. After heating and dissolving the oil, add a surfactant, preservative, antioxidant, and fragrance, and adjust to 70 ° C. This is added to the previous aqueous phase and pre-emulsified. Emulsified particles are homogenized with a homomixer, and deaerated, filtered, and cooled.

Formulation Example 4: Massage cream (O / W type)
Drug: Carbonylation inhibitor (lemon extract) of the present application 0.1% by mass
Oil content: solid paraffin 5.0
Beeswax 10.0
Vaseline 15.0
Liquid paraffin 41.0
Moisturizer: 1,3 Butylene glycol 4.0
Surfactant: glyceryl monostearate 2.0
POE (25) sorbitan monolauric acid 2.0
Esters Alkali: Borax 0.2
Antiseptic: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Purified water: 20.7

Manufacturing method Add water, chemicals, moisturizer, and borax to purified water to prepare an aqueous phase, then heat and adjust to 70 ° C. After heating and dissolving the oil, add a surfactant, preservative, antioxidant, and fragrance, and adjust to 70 ° C. This is gradually added to the previous aqueous phase and pre-emulsified. Emulsified particles are homogenized with a homomixer, and deaerated, filtered, and cooled.

Claims (3)

  A protein carbonylation inhibitor comprising one or more active ingredients selected from olive leaf extract, hydrolyzed pea protein and lemon extract.   The protein carbonylation inhibitor according to claim 1 for preventing or treating a symptom or disease related to carbonylation of a biological protein.   3. The protein carbonylation inhibitor according to claim 2, wherein the symptom or disease related to carbonylation of a biological protein is yellowing of the skin.

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