JP2023076382A - Antiglycative composition - Google Patents

Abstract

To find a highly safe, antiglycative natural product and to provide an antiglycative composition that comprises the found one as an active ingredient.SOLUTION: An antiglycative composition comprises, as an active ingredient, extract from seaweed of Lessoniaceae, Laminariales, and is effective in preventing and improving lifestyle diseases such as diabetes and arteriosclerosis, and various symptoms of aging such as aging-related skin loosening, wrinkles, darkening, cataracts, and osteoporosis.SELECTED DRAWING: None

Description

The present invention relates to techniques for inhibiting anti-glycation.

The saccharification reaction has long been known in the world of food, and it has been shown that heating food causes amino acids and sugars to react, resulting in changes in taste, aroma, and texture. That is, advanced glycation end-products (AGEs) are produced by a non-enzymatic and irreversible reaction (also referred to as browning reaction or Maillard reaction) of amino acids and protein amino groups with reducing sugars.
In recent years, it has become clear that the saccharification reaction of proteins also occurs in vivo, and this has attracted attention. This in vivo saccharification reaction is considered to be a phenomenon in which proteins in the body react with sugars ingested into the body and are denatured. AGEs are produced by repeating chemical reactions.
It is known that AGEs are normally excreted from the body through metabolism. However, it is said that with age, the metabolic rate slows down, accumulating in various tissues in the body and binding to AGEs receptors, causing various symptoms. For example, the accumulation of AGEs in skin tissue contributes to overall skin deterioration.
Collagen is a representative protein present in vivo. Collagen is a protein that is abundant in the body and has a long half-life and a long metabolic cycle. Therefore, it is believed that AGEs are likely to accumulate by reacting with carbohydrates present in the body. In addition, saccharification of collagen causes a cross-linking reaction that cannot normally occur, which is considered to be one of the causes of aging. Glycation of collagen not only causes deterioration of the skin as a whole due to a decrease in skin firmness, but is also thought to be deeply involved in the extension of osteoporosis and osteoarthritis. It helps prevent aging. In addition, it is deeply involved in the development of various diseases such as diabetes and arteriosclerosis, and it is known that AGEs caused by hyperglycemia cause complications such as cataracts, renal dysfunction, and neurosis, especially in diabetic patients. ing. Therefore, it is believed that substances that inhibit the glycation reaction to suppress the production of AGEs and substances that promote the decomposition of the produced AGEs can suppress various age-related diseases and diabetic complications. ing. Therefore, in recent years, amid growing interest in anti-aging, the development of anti-glycation compositions that prevent the accumulation of AGEs in vivo, which can induce various age-related diseases, diabetic complications, and aging phenomena, has been promoted. Desired.

Examples of glycation reaction inhibitors include aminoguanidine (Non-Patent Document 1) and methylguanidine (Patent Document 1). Aminoguanidine and methylguanidine are known to suppress the accumulation of AGEs in tissues and protect against age-related decline in cardiovascular and renal function, but both cause side effects such as liver damage. Its safety is feared because of its potential.
In addition, a search for a safe anti-glycation agent without side effects has been made, and examples of natural antioxidants include kale powder or kale extract (Patent Document 1), an extract of Kabanoanatake (Patent Document 2), and a solvent of mung bean seed coat. extract (Patent Document 3), Budrejaxillaris leaf extract (Patent Document 4), Osmanthus extract (Patent Document 5), and cane extract (Patent Document 6).

Japanese Patent No. 6607418 JP 2019-43887 A JP 2015-182990 A JP 2011-102270 A Japanese Patent No. 5969738 JP 2018-203649 A

Proc. Natl. Acad. Sci. USA, Vol.93, pp.3902-3907, 1996

An object of the present invention is to discover new natural products having anti-glycation activity.

As a result of intensive studies on the above problems, the present inventors have found that an extract of seaweed belonging to the order Laminariaceae and the family Lessoniaceae has an anti-glycation effect, and completed the present invention.
That is, the present invention has the following main configurations.
1. An anti-glycation composition containing an extract of seaweed belonging to the order Laminariaceae, Lessoniaceae, as an anti-glycation active ingredient.
2. 1. Anti-glycation is suppression of production of advanced glycation end products (AGEs). An anti-glycation composition as described.
3. Anti-glycation is protein cross-linking activity,1. An anti-glycation composition as described.
4. 1. The seaweed is kurome. An anti-glycation composition as described.
5. 3. The seaweed is any one of Kurome, Tsuruarame, Sagarame, and Kajime. An anti-glycation composition as described.
6. 2. It is characterized by containing a fraction containing a phenolic substance having relatively high fat solubility and a weakly acidic functional group (for example, a phenolic hydroxyl group, etc.); ~ 5. The anti-glycation composition according to any one of 1.
7. 1. The anti-glycation active ingredient is the ingredient showing the highest activity in the EtOAc layer under acidic and neutral conditions. An anti-glycation composition as described.
8. 7. The main component of the anti-glycation composition showing the highest activity in the EtOAc layer is the 40% MeOH elution fraction; An anti-glycation composition as described.
9. 2. It is characterized by suppressing the production of advanced saccharification products (AGEs). An anti-glycation composition as described.
10. 9. The active ingredient that inhibits the production of advanced glycation end products (AGEs) is 6,8'-bieckol; An anti-glycation composition as described.
11.10. 1. An AGE-derived inflammatory response inhibitor comprising the anti-glycation composition described in .
12. 3. The active ingredient exhibiting protein cross-linking activity is 6,8'-bieckol; An anti-glycation composition as described.
13. An antiglycation agent comprising 6,8'-bieckol as an active ingredient.
14. 13. Any of an agent for suppressing production of advanced glycation end products (AGEs), an activator for severing protein cross-links, and an agent for suppressing inflammatory reaction derived from AGEs. An antiglycation agent as described.
15.1. ~ 5. 7. ~ 10. , 12. 11. The anti-glycation composition according to any one of 11. A pharmaceutical composition containing the inflammatory response inhibitor according to 1.
16.1. ~ 5. 7. ~ 10. , 12. 11. The anti-glycation composition according to any one of 11. A cosmetic composition containing the inflammatory response inhibitor according to 1.
17.1. ~ 5. 7. ~ 10. , 12. 11. The anti-glycation composition according to any one of 11. 3. An anti-glycation seaweed powder containing the inflammatory response inhibitor according to 1 as an active ingredient.
18.1. ~ 5. 7. ~ 10. , 12. 11. The anti-glycation composition according to any one of 11. 3. An anti-glycation food containing the inflammatory response inhibitor according to 1.

1. Solvent extracts of seaweeds belonging to the order Laminariaceae Lessoniaceae, such as kurome, tsuruarame, sagarame, and Ecklonia, show anti-glycation action, which is a mechanism of action newly proposed by the present invention.
2. A pharmaceutical composition containing an anti-glycation composition derived from seaweeds belonging to the order Laminariaceae Lessoniaceae, such as Kurome, Tsuruarame, Sagarame, and Ecklonia, or an anti-glycation seaweed powder containing the same as an active ingredient is a novel functional food. A cosmetic composition containing an anti-glycation composition derived from seaweeds belonging to the order Laminariaceae, Lessoniaceae, Kurome, Tsuruarame, Sagarame, and Ecklonia is a novel functional cosmetic product.
3. Since naturally-derived seaweeds belonging to the order Laminariales, Lessononiaceae, Kurome, Tsuruarame, Sagarame, and Ecklonia have also been eaten, they are highly safe pharmaceutical compositions, cosmetic compositions, or antiglycation agents having antiglycation effects. Seaweed powder can be provided.
4. The anti-glycation composition containing, as an active ingredient, an extract of seaweeds of the present invention, which are seaweeds belonging to the order Laminariaceae, Lessononiaceae, Krome, Tsuruarame, Sagarame, and Ecklonia, has a strong antiglycation effect. It can greatly contribute to the prevention or improvement of various aging phenomena such as habitual diseases, age-related skin sagging, wrinkles, dullness, cataracts, and osteoporosis.
5. The active ingredients exhibiting anti-glycation derived from seaweeds belonging to the order Laminariaceae Lessoniaceae, Kurome, Tsuruarame, Sagarame, and Ecklonia, showed the highest activity in the EtOAc fraction layer, and the main component was present in the 40% MeOH elution fraction.
6. 6,8'-bieckol was identified as the main component of active ingredients exhibiting anti-glycation properties derived from seaweeds belonging to the order Laminariaceae Lessoniaceae, kurome, tsuruarame, sagarame, and Ecklonia.
The 6,8'-bieckol obtained from this seaweed can be used to prevent and treat diabetic complications.
6,8'-bieckol obtained from this seaweed exhibits intraprotein cross-linking activity by cleaving the α-diketone structure, and exhibits AGE production inhibitory activity. It was also suggested that AGEs-based inflammatory reactions could be suppressed.
7. By using 6,8'-bieckol obtained from this seaweed, various aging phenomena such as lifestyle-related diseases such as diabetes and arteriosclerosis, age-related skin sagging, wrinkles, dullness, cataracts, and osteoporosis can be treated. It can greatly contribute to prevention or improvement.

FIG. 2 is a diagram showing results (2) of a production inhibitory activity test for end products of saccharification (AGEs). It is a figure which shows a protein cross-linking activity test result (2). It is a figure which shows the protein cross-linking activity test result of the seaweed of Laminariales, Lessoniaceae. It is a figure which shows the organic-solvent fractionation scheme of the kelp order Lessoniaceae kurome. It is a figure which shows the protein cross-linking activity test result of the organic-solvent fraction of the Lamb order Lessoniaceae. FIG. 2 is a diagram showing the organic solvent fraction of the Kurome MeOH extract. FIG. 2 is a graph showing protein cross-linking activity using the conversion rate of 1-phenyl-1,2-propanedion (PPD) to Benzoic acid as an indicator. Fig. 10 is a graph showing the protein cross-linking activity by PVPP adsorption to the MeOH extract of kurome. FIG. 3 shows reverse phase chromatography of EtOAc layer components. FIG. 10 shows that the main component eluted with 40% MeOH from the EtOAc layer (4.14 g) was separated by HPLC and 206 mg of the main component was obtained. FIG. 11 is a diagram in which the main component in FIG. 10 was determined to be 6,8'-bieckol by structural analysis using 1H-NMR spectrum and 13C-NMR spectrum. 6,8'-bieckol obtained from Kurome shows a protein cross-linking activity equivalent to that of Alageburium (ALT-711), which is known to have an anti-glycation effect. (a) 6,8'-bieckol shows protein cross-linking activity. (b) The protein cross-linking activity of 6,8'-bieckol is equivalent to that of lagebrium (ALT-711). 6,8'-bieckol is a diagram showing the possibility of cleaving intraprotein crosslinks generated by glycation reaction. (a) Quantification method using BSA, (b) 6,8'-bieckol, like the positive control drug ALT-711, cleaves intraprotein crosslinks generated by glycation reaction. FIG. 10 shows. 6,8'-bieckol is a figure which shows the AGE production inhibitory activity. (a) analysis step, (b) 6,8'-bieckol cleaves the α-diketone structure, thereby suppressing the production of AGEs formed on the protein. FIG. 2 shows the effect of AGE production inhibitory protein (6,8'-biecko) on NF-κB activity. (a) Reaction step, (b) Results of measurement of the effect of AGE production inhibitory protein on NF-κB activity.

Embodiments of the present invention will be described below.
Since it was confirmed that the extracts of seaweeds of the present invention, which are seaweeds belonging to the order Laminariaceae, Lessononiaceae, Krome, Tsuruarame, Sagarame, and Eckloniae have an antiglycation effect, the present invention provides a novel seaweed-derived antiglycation composition. It is a thing.
An anti-glycation composition containing, as active ingredients, an extract of kurome, a seaweed belonging to the family Lessoniaceae of the order Laminariales, an extract of tuna lame, an extract of sarcophagus, and an extract of Ecklonia japonicum, has an anti-glycation effect and is therefore useful for treating diabetes and arteriosclerosis. It can greatly contribute to the prevention or improvement of various aging phenomena such as lifestyle-related diseases such as sclerosis, age-related skin sagging, wrinkles, dullness, cataracts, and osteoporosis.
The active ingredients exhibiting anti-glycation derived from seaweeds belonging to the order Laminariaceae Lessoniaceae, Kurome, Tsuruarame, Sagarame, and Ecklonia, showed the highest activity in the EtOAc fraction layer, and the main component was present in the 40% MeOH elution fraction. It was possible to identify 6,8'-bieckol as the main component of the active ingredient showing antiglycation. The 6,8'-bieckol obtained from this seaweed can be used to prevent and treat diabetic complications.
6,8'-bieckol obtained from this seaweed exhibits intraprotein cross-linking activity by cleaving the α-diketone structure, and exhibits AGE production inhibitory activity. In addition, there is a possibility that AGEs-based inflammatory reactions can be suppressed.
Pharmaceutical compositions, cosmetic compositions, including anti-glycation compositions containing, as active ingredients, extracts of kuromae, extracts of trumpet, extracts of ragweed, and extracts of Ecklonia, which are expected to have such a mechanism of action, Alternatively, anti-glycation seaweed powder containing it as an active ingredient provides new functional foods, pharmaceuticals, and cosmetics.
By using 6,8'-bieckol obtained from this seaweed, various aging phenomena such as lifestyle-related diseases such as diabetes and arteriosclerosis, age-related skin sagging, wrinkles, dullness, cataracts, and osteoporosis can be treated. It can greatly contribute to prevention or improvement.

The anti-glycation composition of the present embodiment contains, as active ingredients, an extract of Kurome, an extract of Tsuruarame, an extract of Sagarame, and an extract of Ecklonia.
Here, the "extract" in the present embodiment means an extract obtained by using the seaweed as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or any of these Both crude and purified products are included.

<Production of Kurome extract, Tsuruarame extract, Sagarame extract, and Ecklonia extract>
In the present embodiment, the raw materials for extraction used to produce the black bream extract, the crane arame extract, the crocodile extract, and the Ecklonia extract are Kurome (scientific name: Ecklonia kurome), Crane arame (scientific name: Ecklonia stolonifera), and Sagarame (scientific name : Eisenia arborea) and Kajime (scientific name: Ecklonia cava).

Ecklonia kurome (black cloth) is a brown alga belonging to the order Laminariaceae and the family Lessoniaceae. Distributed in the central to southern Pacific coast of Honshu, Shikoku, Kyushu, the Seto Inland Sea, and the Japan coast of Honshu, inhabiting the subtidal zone. The size is 1 to 2 m in height.
The body consists of a stem and a membranous leaf, and several lateral leaves extend from the leaf. It looks a lot like Ecklonia japonica but is a little smaller, and can be distinguished by the uneven wrinkles on the entire leaf and the fact that the center of the leaf is not thick. However, there are many variations in form.

Ecklonia stolonifera is a brown algae of the order Laminariaceae and the family Lessoniaceae. It is distributed from the southern part of Hokkaido, the Japanese coast of Honshu to the northern coast of Kyushu. The size is 25 to 150 cm in height.
The body consists of a stem and a membranous leaf. In addition, the leaves are wrinkled throughout. It is characterized by creeping branches extending from the base and new leaves appearing from the tip, and the name ``vine'' comes from the creeping branches.

Eisenia arborea (Sagara cloth) is a brown alga belonging to the order Laminariales and the family Lessoniaceae. A seaweed similar to arame, it is distributed from Omaezaki in Shizuoka Prefecture to the Kii Peninsula and Tokushima Prefecture. The side leaves of Arame are often branched, whereas the side leaves of Sagarame are hardly branched.

Ecklonia cava (Ecklonia cava) is a brown alga belonging to the order Laminariaceae and the family Lessoniaceae. It is distributed in the central Pacific coast of Honshu, the Seto Inland Sea, the southern part of the Japanese coast of Honshu, and Kyushu, and inhabits from the low tide line to the subtidal zone at a depth of 20m. The size is 1 to 3 m in height.
The sporophyte consists of a short stem and an elliptical leaf part in juveniles, and the stem grows as it grows. Lateral leaves may be further branched. The leaf part is smooth without wrinkles and hard to the touch. Color is brown to dark brown. They live in colonies called marine forests.

As used herein, the terms "kurome", "tsuruarame", "sagarame", and "kajime" refer to the entire algal body of kurome or tsuruarame, sagarame, and elk. Contains roots. Further, in the present specification, the seaweed belonging to the "Laminariales, Lessoniniaceae" includes arame (scientific name: Eisenia bicyclis) and antokume (scientific name: Eckloniopsis radicosa). These seaweeds are not common, but have local edible experience.

Although the details of substances having anti-glycation action contained in each of the extract of Japanese bream, the extract of Tulu arame, the extract of Sagarame and the extract of Ecklonia are unknown, the extraction method generally used for extracting seaweed An extract having this effect can be obtained from the seaweed by the method.

For example, after drying the seaweed, it can be obtained as it is or by pulverizing it with a pulverizer and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun, or may be performed using a commonly used dryer. In addition, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a nonpolar solvent such as hexane. By performing a pretreatment such as degreasing, the extraction treatment of the seaweed with a polar solvent can be performed efficiently.
As the extraction solvent, it is preferable to use a polar solvent, such as water and hydrophilic organic solvents, which are used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent. is preferred.
Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, deep sea water, and water subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic adjustment, buffering, and the like. Therefore, water that can be used as an extraction solvent in the present embodiment includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, and the like.
Hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

When a mixture of two or more polar solvents is used as the extraction solvent, the mixture ratio can be appropriately adjusted. For example, when using a mixture of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of a lower aliphatic alcohol with 10 parts by volume of water, and water and a lower aliphatic ketone. When using a mixture, it is preferable to mix 1 to 40 parts by volume of lower aliphatic ketone with 10 parts by volume of water, and when using a mixture of water and polyhydric alcohol, 10 parts by volume of water It is preferable to mix 10 to 90 parts by volume of polyhydric alcohol per part.

The extraction treatment is not particularly limited as long as the soluble components contained in the raw material for extraction can be eluted into the extraction solvent, and can be carried out according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent of 5 to 15 times the amount (mass ratio) of the extraction raw material, the soluble component is extracted at room temperature or under reflux heating, and then filtered to remove the extraction residue. You can get the liquid. A paste-like concentrate is obtained by distilling off the solvent from the obtained extract, and a dried product is obtained by further drying the concentrate.
The extract obtained as described above can be used as it is as an active ingredient of the anti-glycation composition, but it is easier to use as a concentrate or dried product.
In addition, since the extract from the seaweed has a unique odor, it is possible to carry out purification for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in physiological activity. Since it is not used in a large amount when it is blended into a liquid, there is practically no problem even if it is left unrefined.

<Anti-glycation composition>
Each seaweed extract obtained as described above has an anti-glycation effect. As shown in Tables 2 and 3 and FIGS. 1 to 3, the anti-glycation action of the kurome extract of the present invention, or the monolithic cricket extract, the rattlesnake extract, or the Ecklonia japonicus extract is stronger than that of other seaweed extracts. Therefore, by utilizing this action, it can be used as an active ingredient of anti-glycation composition. Furthermore, as shown in FIG. 5, a fraction having relatively high fat solubility and containing a weakly acidic functional group (eg, phenolic hydroxyl group, etc.) can also be used as an active ingredient of the antiglycation composition.
Such anti-glycation compositions can be incorporated into pharmaceutical compositions, cosmetic compositions and the like. Alternatively, it can be used as an anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient.

The anti-glycation composition containing the extract of black bream, the extract of the trumpet, the extract of the rattlesnake, and the extract of the Ecklonia japonicus is a raw material for pharmaceutical compositions, cosmetic compositions, or anti-glycation seaweed containing it as an active ingredient. It can be used as a powder. An anti-glycation composition containing an extract of black bream, an extract of trumpet cricket, an extract of rattlesnake, an extract of Ecklonia japonicus is used in pharmaceutical compositions (e.g. oral tablets, capsules) and cosmetic compositions (e.g. , lotions for application, creams).
These pharmaceutical compositions and cosmetic compositions are particularly useful for prevention of various aging phenomena such as lifestyle-related diseases such as diabetes and arteriosclerosis, age-related skin sagging, wrinkles, dullness, cataracts and osteoporosis. Or it can be used for improvement. In addition, an anti-glycation composition containing an extract of black bream, an extract of trumpet cricket, an extract of ragweed, and an extract of Ecklonia japonicus can be used as an active ingredient of seaweed powder for anti-glycation, for example, as a health food such as an oral supplement. In particular, it is used to prevent or improve various aging phenomena such as lifestyle-related diseases such as diabetes and arteriosclerosis, age-related skin sagging, wrinkles, dullness, cataracts, and osteoporosis. can be

Since the anti-glycation composition of the present embodiment has an anti-glycation effect and is safe, it is suitable for formulation as a pharmaceutical composition, for example. The antiglycation composition of the present invention can be made into a form such as a tablet and used as a pharmaceutical composition.
In addition, the pharmaceutical composition of the present invention may contain additives or pharmaceutically acceptable carriers other than the antiglycation agent of the present invention. Examples of such additives or pharmaceutically acceptable carriers include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, Suspending agents, emulsifiers, solvents, thickeners, mucolytic agents, wetting agents, preservatives and the like.

Although the form of the pharmaceutical composition of the present invention is not particularly limited, it may be either an oral formulation or a parenteral formulation. Oral preparations include granules, powders, tablets, capsules, syrups, tinctures, jellies and the like. Parenteral agents include injections, drops, ointments, nasal drops, suppositories and the like.
The dosage of the pharmaceutical composition of the present invention is not particularly limited. Dosages can be in the range of ~100 mg/kg body weight, preferably 1-50 mg/kg body weight. In addition, when the pharmaceutical composition of the present invention is parenterally administered, the content is 0.05 mg/kg body weight to 50 mg/kg body weight, preferably 0.5 mg/kg body weight to 50 mg/kg body weight. can do.

Since the anti-glycation composition containing the extract of black bream, the extract of tuna cricket, the extract of rattlesnake, and the extract of Ecklonia japonicum has anti-glycation effect and is safe and pleasant to use when applied to the skin, For example, it is suitable for blending as external cosmetics. The anti-glycation composition of the present invention can be used as an external skin preparation in the form of ointments, creams, gels, lotions, milky lotions, packs, poultices, bath preparations, eye drops, nose drops, and the like. can be done.

The anti-glycation composition of the present invention can be incorporated into skin cosmetics, shampoos, rinses, conditioners and other hair cosmetics. be able to. Furthermore, various adjuvants can be added for the purpose of enhancing or supplementing the functions of the external preparation of the present invention. For example, base materials include glycerol, ethanol, paraben, butylene glycol, and the like.

Additives include excipients (silicon-based polymers), fragrances, pigments, preservatives (parabens, etc.), thickeners (silicon-based polymers, acrylic polymers, carboxyvinyl-based polymers, etc.), chelating agents (EDTA, etc.). , sweeteners (sucralose, etc.), cooling agents (menthol, etc.), preservatives (phenoxyethanol, etc.), and the like.

Examples of auxiliary agents include other medicinal ingredients and oils (unsaturated fatty acids such as linoleic acid, linolenic acid, palmitic acid, DHA, EPA and their derivatives, linseed oil, coconut oil, jojoba oil, olive oil, squalane, Oils extracted from animals and plants such as squalene, horse oil, rice bran oil, castor oil and their derivatives, etc.), moisturizing agents (collagen or its decomposition products, collagen-like peptides including carrot extract, soybean peptides, amino acids, hyaluronic acid such as mucopolysaccharides, amino acids such as chondroitin, sugars such as trehalose, seaweeds, surfactants (lecithin, fatty acid esters, amino acid derivatives, etc.), ultraviolet absorbers (zinc oxide, titanium oxide, etc.), absorption promoters, etc. mentioned.

When using the anti-glycation composition of the present invention, the amount to be blended can be appropriately determined depending on the combination with other ingredients and the form of the composition. Generally, it is preferably 0.00001% by weight or more, more preferably 0.001% by weight or more in terms of dry weight in the total compounding amount.

The anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient is an anti-glycation composition containing an extract of kurome, an extract of tsuruarame, an extract of purslane and an extract of Ecklonia japonicum, as well as glucose, fructose, Sucrose, maltose, maltitol, sorbitol, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, casein, gelatin, pectin, agar, amino acids, excipients, fillers, binders, thickeners, Emulsifiers, coloring agents, flavoring agents, food additives, seasonings, preservatives and the like may further be contained as appropriate. Such anti-glycation seaweed powder can be formed into forms such as powder, granules, capsules, tablets, syrups, suspensions, etc., and can also be processed into candy, etc., depending on the application. For example, an oral supplement containing the anti-glycation seaweed powder can be produced by a method commonly used by those skilled in the art.
The amount of the anti-glycation composition to be added to the oral supplement, the method of blending, and the time of blending can be appropriately selected. Oral supplements may contain the anti-glycation composition in an amount of 0.1-100% by weight, more preferably 10-80% by weight (extract basis) of the total formulation composition.

The anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient can be ingested as it is, or it can be ingested after being dissolved or suspended in a solvent such as water. The anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient can be orally ingested before, during or after meals. In addition, the anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient can be added to food and drink, and then eaten.
Foods to which the anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient is added include, for example, home-use diabetic diet, liquid diet, food for the sick (combination food for diabetic diet preparation, etc.), and food for specified health use. Examples include, but are not limited to, foods, diet foods, or foods and drinks containing carbohydrates as main ingredients.
Specific food forms include, but are not limited to, rice products, barley products, vegetable products, dairy products, and soft drinks.

The addition or processing of the anti-glycation seaweed powder containing the anti-glycation composition as an active ingredient to food or drink can be carried out by a method commonly used by those skilled in the art, and the amount, method and time of incorporation can be appropriately selected. can. It can also be added to feeds for animals other than humans, such as livestock or pets (eg, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.).

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

(Preparation of seaweed extract)
23 types of seaweed (Eurhemum muricatum, Mahnori, Hijiki, Wakame (leaf), Wakame (stem), Tsunomata (yellow), Mozuku, Tosaka nori (blue), Tosaka nori (white), Wakame (spore leaf part), Tsunomata (blue) ), Tsunomata (red), Tosaka nori (red), Matsu nori, Hosobanosakamodoki, Asakusa nori, Suginori (red), Funori, Suginori (green), Kombu, Kurome, Uppuruinori, Gracilaria) were prepared.

3 g of each was ground in a coffee grinder and collected in a 50 mL plastic tube.
Then, 30 mL of methanol was added to the plastic tube, and ultrasonic extraction was performed three times for 15 minutes. After centrifuging the plastic tube at 15,000 rpm for 5 minutes, the supernatant was concentrated under reduced pressure to obtain 23 types of seaweed methanol extracts.
The obtained 23 types of seaweed extracts were dissolved in 600 μL of dimethylsulfoxide (DMSO) and used as evaluation samples for the following tests.

(Glycation end product (AGEs) production inhibitory activity test)
After preparing the reaction solutions shown in Table 1 below, they were incubated at 60° C. for 30 hours.

After cooling once in a refrigerator after completion of the incubation, 100 μL of the reaction solution was transferred to a 1.5 mL plastic tube, and 10 μL of 100% trichloroacetic acid (hereinafter referred to as TCA) was added and stirred. After stirring, centrifugation was performed at 15,000 rpm for 4 minutes at 4° C., the supernatant was removed with an aspirator, and the precipitate was redissolved in 400 μL of alkaline phosphate buffer (PBS) (−) (pH 10). 200 μL of the redissolved solution was placed in the wells of a 96-well plate for fluorescence measurement (manufactured by DYNEX), and the fluorescence intensity was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm to determine the “apparent inhibitory effect”.

In addition, DMSO was added instead of the evaluation sample, and incubated at 60° C. for 30 hours in the same manner as described above. 50 μL of the sample for evaluation was added to 100 μL of the incubated reaction solution and well stirred. After that, 10 μL of 100% TCA was added, stirred, and centrifuged at 15,000 rpm and 4° C. for 4 minutes. After removing the supernatant, the precipitate was redissolved with 400 μL of alkaline PBS(-) (pH 10), and fluorescence intensity was measured. The "quenching effect" was determined by comparing the fluorescence intensities.

By subtracting the "quenching effect" from the "apparent inhibitory effect", the true AGE production inhibitory activity was calculated.

<Protein cross-linking activity test>
Phosphate buffer (pH 7.4) 800 μL (-) (pH 10) 400 μL, 100 mM PTB 100 μL (-) (pH 10) 400 μL, 100 mM 1-phenyl-1,2-propanedion 100 μL, 50 mM benzoic acid 100 μL, H20 100 μL, for evaluation 100 μL of the sample (23 types of seaweed extract) solution was added to the Eppendorf tube to bring the total volume to 1 mL.
After stirring, the reaction was carried out in a bioshaker at 37°C for 8 hours. Then 20 μL of 2N HCl was added to stop the reaction. After stirring, centrifugation was performed at 15,000 rpm and 4° C. for 5 minutes, and the supernatant was subjected to high performance liquid chromatography (HPLC) as an analytical sample. The protein cross-linking activity was calculated from the area area of the peak corresponding to Benzoic acid obtained by HPLC.

(Conditions for analysis of Benzoic acid by HPLC)
・Column: Mightysil RP-18 (Φ150×2mm, Kanto Chemical Industry, Lot No.8022371)
・Flow rate: 1 mL/min
・Detection wavelength: UV 250 nm
・ Solvent: 40% methanol / 0.1% trifluoroacetic acid (TFA)
・Temperature: 40℃
・Injection volume: 10 μL

Table 2 and FIG. 1 show the results of the production inhibitory activity test for advanced glycation end products (AGEs).
As shown in Table 2, 20 to 40% of weak AGEs are generated in Uppulu-no-Nori of the family Cathophyllaceae, Asakusanori of the family Cathophyllaceae of the order Cathoeniformes, Tosaka nori (red) of the family Myrrhidae of the order Coleoptera, and Tosakamodoki of the family Calyptogenyaceae. The inhibitory activity was shown to be 41 to 70% of the moderate AGE production inhibitory activity in the leaves and stems of wakame seaweed belonging to the order Laminariaceae, and the Mozuku belonging to the family Nagamatsumo, of the order Laminariaceae of the present invention. has a very strong AGE production inhibitory activity of 97%.

Kurome, which had the strongest AGE production inhibitory activity in Table 2, and Mozuku, which had the second strongest AGE production inhibitory activity, were further examined in detail while changing the concentration of the seaweed extract. As shown in the production inhibitory activity test results (2)), the AGEs production inhibitory activity of Mozuku, which is a seaweed belonging to the family Arboraceae of the order Nagamatsumo, was about 60% even at the highest concentration of 10.0%. The kurome, which is a seaweed belonging to the order Laminariaceae Lessoniaceae of the invention, has a high AGE production inhibitory activity of 50% or more even at a low concentration of 0.3% (v/v).

Next, Table 3 and FIG. 2 show the results of the protein cross-linking activity test.
As shown in Table 3, Hijiki (bud) of the Fucusaceae Sargassum family, Wakame (stem) of the Laminariaceae Lambaceae family, Mozuku of the Nagamatsumo order Nagamatsumo family, Tosakanori of the Cucurbitaceae Myrrhidae (blue), and Bovine cattle family Bollaceae The kelp of the present invention showed a weak protein cross-linking activity of 10 to 20% for the kelp of the present invention, a weak 10 to 20% for the Gracilaria of the Gracilaria, and a moderate 44% of the protein cross-linking activity for the wakame (spore leaf part) of the Lambs, Lambs. Kurome of the order Lessoniaceae has a very strong protein cross-linking activity of 87%.

Further detailed examination was carried out while changing the concentration of the kurome extract, which had the strongest AGEs cross-linking activity in Table 2. As shown in FIG. 2 (protein cross-linking activity test results (2)), Kurome, which is a seaweed belonging to the order Laminariaceae of the present invention and belonging to the family Lessoniidae, has a protein cross-linking activity of about 20% even at a low concentration of 1.25% (v/v).

As can be seen from Table 2 and FIG. 1, Table 3 and FIG. 2, in both the production inhibitory activity of advanced saccharification end products (AGEs) and the protein cross-linking activity, crome of the Order Laminariaceae Lessoniaceae has a strong activity. Therefore, it was shown that kurome, a seaweed belonging to the family Lessoniaceae of the order Laminaria, has an anti-glycation effect.

(Preparation of Seaweed Extract of Laminariales, Lessoniaceae)
3 g of each of the dried seaweeds (Kurome, Tsuruarame, Sagarame, Ecklonia) of the order Laminariaceae and the family Lessoniaceae were pulverized in a coffee mill and collected in a 50 mL plastic tube. Then, 30 mL of methanol was added to the plastic tube, and ultrasonic extraction was performed three times for 15 minutes. 15,000 r.p.m. p. After centrifugation at 10 m for 5 minutes, the supernatant was concentrated under reduced pressure to obtain a methanol extract of seaweed belonging to the order Laminariaceae, Lessoniaceae. The resulting seaweed extract belonging to the order Laminariaceae Lessoniaceae was dissolved in 600 μL of dimethylsulfoxide (DMSO) and used as an evaluation sample for the following tests.

(Protein cross-linking activity test of seaweeds of Laminariales, Lessoniaceae)
(Protein cross-linking activity test)
Phosphate buffer (pH 7.4) 800 μL, 100 mM PTB 100 μL, 100 mM 1-phenyl-1,2-propanedion 100 μL, 50 mM benzoic acid 100 μL, H20 100 μL, sample for evaluation (23 types of seaweed extract) solution 100 μL Eppendorf Added to the tube to bring the total volume to 1 mL.

After stirring, the reaction was carried out in a bioshaker at 37°C for 8 hours. Then 20 μL of 2N HCl was added to stop the reaction. After stirring, centrifugation was performed at 15,000 rpm and 4° C. for 5 minutes, and the supernatant was subjected to high performance liquid chromatography (HPLC) as an analytical sample. The protein cross-linking activity was calculated from the area of the peak corresponding to Benzoic acid obtained by HPLC.

<Conditions for analysis of Benzoic acid by HPLC>
・Column: Mightysil RP-18 (Φ150×2mm, Kanto Chemical Industry, Lot No.8022371)
・Flow rate: 1 mL/min
・Detection wavelength: UV 250 nm
・ Solvent: 40% methanol / 0.1% trifluoroacetic acid (TFA)
・Temperature: 40℃
・Injection volume: 10 μL

As shown in FIG. 3 (results of protein cross-linking activity test of seaweed of the order Laminariaceae Lessoniaceae), it has a protein cross-linking activity of 70% or more in seaweeds of the Laminariaceae Lessoniaceae family, such as Kurome, Tsuruarame, Sagarame, and Eckloniae. Therefore, it has an anti-glycation effect.

<Protein cross-linking activity test of organic solvent fraction of seaweed of Laminariales Lessoniaceae>
For the purpose of clarifying the polarity (solvent transfer property) of the substance having activity, the methanol extract of kuromae was subjected to organic solvent fractionation as shown in FIG. Each fraction was evaluated for protein cross-linking activity.

That is, 3 g of dried Kurome was collected in 50 mL plastic tubes, and four tubes were prepared. 30 mL of methanol was added and ultrasonic extraction was performed three times for 15 minutes. After centrifugation at 15,000 rpm for 5 minutes, the supernatant was concentrated under reduced pressure to obtain an extract. 10 mL each of water of pH 3, 7, and 10 was added to each of the three extracts obtained, and 10 mL of chloroform (CHCl3) was further added. After stirring with a vortex mixer to completely dissolve the extract, the mixture was centrifuged at 10,000 rpm for 5 minutes to obtain a CHCl3 layer. This operation was performed once more to obtain a CHCl3 extract. Each extract was obtained by extracting twice with ethyl acetate (EtOAc) and 10 mL of water-saturated butanol (n-BuOH). The obtained extract was concentrated under reduced pressure to perform organic solvent fractionation at each pH.

The resulting extract was dissolved in 600 μL of DMSO and used for evaluation as a protein cross-linking activity evaluation solution. As a result, at pH 3 and 7, high activity was observed in the CHCl3 extract. Under the same conditions, activity was also observed in the ethyl acetate extract. At pH 10, no activity was observed in any fraction. From this result, it is suggested that the chemical structure of the active substance contained in kurome, a seaweed belonging to the order Laminariaceae of the present invention, has relatively high fat solubility and the presence of weakly acidic functional groups (for example, phenolic hydroxyl groups, etc.). rice field.
On the other hand, under alkaline conditions, no activity was observed in any of the fractions, indicating that the active principal compound with protein cross-linking activity is unstable under alkaline conditions, and this applies to many phenolic substances. It is assumed that the active main compound contained in the seaweed Kurome of the present invention, Laminariales, Lessoniaceae, is a phenolic substance.

(Manufacturing of tablets)
Using the extract obtained in Example 2, tablets having the following composition were produced according to a conventional method.
Tsuruarame extract combination tablet (Composition) (Composition: weight %)
Tsuruarame extract 24
Lactose 63
Cornstarch 12
guar gum 1

(manufacture of juice)
A juice having the following composition was produced in accordance with a conventional method using the extract obtained in Example 2.
Kurome extract-containing juice (Composition) (Formulation: wt%)
Frozen Concentrated Satsuma Mandarin Juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Perfume 0.2
Pigment 0.1
Kurome extract 0.2
water 83.28

(Manufacture of face cream)
Using the extract obtained in Example 2, a face cream having the following composition was produced according to a conventional method.
Face cream containing Sagarame extract (Composition) (Formulation: weight %)
Isopropyl isostearate 8.0
Jojoba oil 6.0
Cetanol 8.0
Stearyl alcohol 2.0
Polyoxyethylene lauryl ether 1.5
Propylene glycol 6.0
Sorbitol 1.0
Paraben 0.4
Sagarame extract 0.5
Vitamin E 0.5
Perfume 0.1
Purified water 66.0

The anti-glycation composition containing the extract of seaweed belonging to the order Laminariaceae of the present invention as an active ingredient has the activity of suppressing the production of advanced glycation end products (AGEs) and the activity of cleaving protein cross-links, and is therefore useful for diabetes and arteriosclerosis. and other lifestyle-related diseases, age-related skin sagging, wrinkles, dullness, cataracts, osteoporosis, and other various aging phenomena.

Furthermore, a test was conducted to identify the active ingredient contained in the extract of seaweed belonging to the order Laminariaceae and the family Lessoniaceae.
Since the test method for identifying the anti-glycation component described below and the mechanism of action is conventionally well-known, the detailed description is omitted and the main items are described.

(Anti-glycation ingredient search test 1: Organic solvent fractionation test)
FIG. 6 shows the organic solvent fraction of the Kurome MeOH extract. FIG. 7 is a graph showing the protein cross-linking activity using the conversion rate of 1-phenyl-1,2-propanedione (PPD) to Benzoic acid as an index.
The obtained MeOH extract of Kurome was subjected to liquid-liquid partitioning with chloroform/water (CHCl3/H2O) (pH 3, 7 or 10). After that, ethyl acetate (EtOAc) was added to the aqueous layer, and liquid-liquid partitioning was performed in the same manner. Furthermore, butanol (n-BuOH) was added to the aqueous layer to perform liquid-liquid partitioning (see FIG. 6).
As a result, as shown in FIG. 7, the highest activity was observed in the EtOAc layer under acidic and neutral conditions. Using 1-phenyl-1,2-propanedione (PPD) as a protein cross-linking model compound, benzoic acid produced by cleavage of the α-diketone structure in the PPD structure was quantified by high performance liquid chromatography (HPLC). Assess the cross-linking activity. Higher conversion to benzoic acid indicates stronger cross-linking activity.

(Anti-glycation component search test 2: PVPP adsorption test)
FIG. 8 is a graph showing the protein cross-linking activity by PVPP adsorption to the MeOH extract of kurome.
The MeOH extract of Kurome has a concentration-dependent protein cross-linking activity. However, when Polyvinylpolypyrrolidone (PVPP), a polyphenol-specific adsorption resin, was added to the MeOH extract of Kurome, the protein cross-linking activity disappeared (see FIG. 8).
From this, it is inferred that the protein cross-linking scission active substance in kurome is a polyphenolic compound.

(Anti-glycation component search test 3: reversed-phase chromatography analysis)
FIG. 9 shows reverse phase chromatography of the EtOAc layer components.
The EtOAc layer showing the highest activity shown in FIG. 7 in the fractions of Test 1 (FIG. 6) was subjected to reverse phase chromatography using water/methanol (H ₂ O/MeOH) solutions of various ratios. As a result, the main component was obtained in the 40% MeOH elution fraction as shown in FIG.

(Anti-glycation component search test 4: isolation test)
Fig. 10 shows that the main component eluted from 40% MeOH by reversed-phase chromatography from the EtOAc layer (4.14 g) was separated by HPLC to obtain 206 mg of the main component.
FIG. 11 is a diagram showing that the main component in FIG. 10 was determined to be 6,8′-bieckol by structural analysis using 1H-NMR spectrum and 13C-NMR spectrum.
From the EtOAc layer (4.14 g) where the highest activity was observed, the main component eluted with 40% MeOH by reversed-phase chromatography was fractionated by HPLC to obtain 206 mg of the main component. Structural analysis was performed on this main component using 1H-NMR spectrum and 13C-NMR spectrum, and the main component was determined to be 6,8'-bieckol (reverse phase chromatography shown in FIG. 10, (see structural analysis shown).

(Anti-glycation component search test 5: protein cross-linking activity test)
Fig. 12 shows that 6,8'-bieckol obtained from Kurome has protein cross-linking activity equivalent to that of Alageburium (ALT-711), which is known to have anti-glycation activity. Figure 12(a) shows that 6,8'-bieckol exhibits protein cross-linking activity. Figure 12(b) shows that the proteolytic cross-linking activity of 6,8'-bieckol is comparable to lagebrium (ALT-711).
6,8'-bieckol obtained from Kurome had a protein cross-linking activity equivalent to that of Alageburium (ALT-711), which is known to have an anti-glycation effect (see FIG. 12(b)). However, phloroglucinol and resorcinol, which are structural units of 6,8'-bieckol, did not show any activity, suggesting that the three-dimensional structure of 6,8'-bieckol is important for expression of activity. (See FIG. 12(b)).

(Anti-glycation component search test 6: Intraprotein crosslink cutting test)
Figure 13 is a diagram showing the possibility that 6,8'-bieckol cleaves the intraprotein crosslinks generated by the glycation reaction. b) shows the possibility that 6,8'-bieckol, like the positive control drug ALT-711, cleaves intraprotein crosslinks generated by glycation reaction.
After Collagen was immobilized on a 96-well microplate, AGEs-BSA was bound.
Then, ALT-711 or 6,8'-bieckol was added, and remaining BSA was quantified using an anti-BSA antibody (see Fig. 13(a)).
As a result, it was suggested that 6,8'-bieckol, like ALT-711, which is a positive control drug, may cleave intraprotein crosslinks generated by glycation reaction (see FIG. 13(b)).

(Anti-glycation component search test 7: AGE production inhibitory activity test)
FIG. 14 is a diagram showing the AGE production inhibitory activity of 6,8′-bieckol. Fig. 14(a) shows the analysis process, and Fig. 14(b) shows that 6,8'-bieckol cleaves the α-diketone structure to suppress the production of AGEs formed on the protein.
4 mg/ml BSA (bovine serum albumin) and 20 mM glucose were added to 50 mM phosphate buffer (pH 7.4) and reacted at 60° C. for 48 hours. After that, trichloroacetic acid was added and centrifuged at 15,000 rpm for 3 minutes. After dissolving the obtained precipitate in alkaline PBS buffer, the amount of AGEs produced was measured using a fluorometer (excitation wavelength 360 nm). , fluorescence wavelength 460 nm) (see FIG. 14(a)).
As a result, 6,8'-bieckol inhibited the formation of AGEs formed on the protein by cleaving the α-diketone structure, similar to the positive control drug aminoguanidine (see Fig. 14(b)). .

(Anti-glycation component search test 8: Effect test on NF-κB activity of AGE production inhibitory protein)
FIG. 15 shows the effect of AGE production inhibitory protein (6,8′-biecko) on NF-κB activity. FIG. 15(a) shows the reaction process, and FIG. 15(b) shows the results of measurement of the effect of AGE production inhibitory protein on NF-κB activity.
BSA and glucose were added in the presence of 6,8'-bieckol or aminoguanidine and allowed to react at 37°C for 3 months. After the reaction, AGEs-BSA was produced and added to human embryonic kidney cells 293 (HEK 293 cells) in which pNF-kB-Luc was expressed. The effect on κB activity was measured (see FIG. 15(a)).
As a result, a decrease in NF-κB activity was observed in BSA in which AGEs production was suppressed by 6,8'-bieckol.
From the above, it was suggested that 6,8'-bieckol, like aminoguanidine, which is a positive control drug, suppresses the formation of AGEs due to protein glycation reaction and suppresses the inflammatory reaction based on AGEs.

Claims (18)

An anti-glycation composition containing an extract of seaweed belonging to the order Laminariaceae, Lessoniaceae, as an anti-glycation active ingredient. 2. The anti-glycation composition according to claim 1, wherein the anti-glycation is suppression of production of advanced glycation end products (AGEs). 2. The anti-glycation composition of claim 1, wherein the anti-glycation is protein cross-linking activity. 3. The anti-glycation composition according to claim 2, wherein the seaweed is Kurome. 4. The anti-glycation composition according to claim 3, wherein the seaweed is Kurome, Tsuruarame, Sagarame or Ecklonia. The antiglycation composition according to any one of claims 2 to 5, characterized by containing a fraction containing a phenolic substance having relatively high fat solubility and a weakly acidic functional group. 2. The anti-glycation composition according to claim 1, wherein the anti-glycation active ingredient is the ingredient showing the highest activity in the EtOAc layer under acidic and neutral conditions. 8. The anti-glycation composition according to claim 7, wherein the main component of the anti-glycation composition showing the highest activity in the EtOAc layer is the 40% MeOH elution fraction. 3. The anti-glycation composition according to claim 2, which inhibits the production of advanced glycation end products (AGEs). 10. The anti-glycation composition according to claim 9, wherein the active ingredient that inhibits the production of advanced glycation end products (AGEs) is 6,8'-bieckol. 11. An AGEs-derived inflammatory response inhibitor comprising the anti-glycation composition according to claim 10. 4. The anti-glycation composition according to claim 3, wherein the active ingredient exhibiting protein cross-linking activity is 6,8'-bieckol. An antiglycation agent comprising 6,8'-bieckol as an active ingredient. 14. The anti-glycation agent according to claim 13, which is an agent for suppressing production of advanced glycation end products (AGEs), an agent for activating protein cross-linking, or an agent for suppressing inflammatory reaction derived from AGEs. A pharmaceutical composition containing the anti-glycation composition according to any one of claims 1 to 5, 7 to 10 and 12 or the inflammatory response inhibitor according to claim 11. A cosmetic composition containing the anti-glycation composition according to any one of claims 1 to 5, 7 to 10 and 12 or the inflammatory response inhibitor according to claim 11. An anti-glycation seaweed powder comprising the anti-glycation composition according to any one of claims 1 to 5, 7 to 10 and 12 or the inflammatory reaction inhibitor according to claim 11 as an active ingredient. An anti-glycation food containing the anti-glycation composition according to any one of claims 1 to 5, 7 to 10 and 12 or the inflammatory response inhibitor according to claim 11.

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