JP2023025533A - Precursor fat cell differentiation inhibitor, beverage/food product, pharmaceutical and cosmetic product containing the precursor fat cell differentiation inhibitor, and method of preventing or remedying obesity or metabolic syndrome - Google Patents

Abstract

To provide a differentiation inhibitor that inhibits differentiation of a precursor fat cell to a fat sell; a beverage/food product, a pharmaceutical, and a cosmetic product for anti-metabolic syndrome including the differentiation inhibitor; and a method of preventing or remedying metabolic syndrome.SOLUTION: A precursor fat cell differentiation inhibitor includes melanin as an active principle, preferably including melanin and melanoprotein derived from squid ink, and particularly preferably including melanin derived from squid ink subjected to enzyme processing and/or melanoprotein subjected to enzyme processing.SELECTED DRAWING: None

Description

The present technology relates to preadipocyte differentiation inhibitors. Specifically, the present technology includes a preadipocyte differentiation inhibitor, an anti-obesity or anti-metabolic syndrome food and drink containing the preadipocyte differentiation inhibitor, an anti-obesity or anti-metabolic syndrome drug, and an anti-obesity or The present invention relates to an anti-metabolic syndrome cosmetic and a method for preventing or treating obesity or metabolic syndrome.

Adipocytes are the major cell type present in adipose tissue. Cells that develop from mesodermal pluripotent stem cells and differentiate into mature adipocytes via lipoblasts, preadipocytes, immature adipocytes, and white adipocytes that are responsible for fat synthesis, storage and release, and adipocytes. It is classified as brown fat cells that decompose and generate heat.

Obesity is considered a risk factor for diabetes, dyslipidemia, hypertension, fatty liver, sleep apnea syndrome, and various cancers. In addition, obesity is strongly associated with enlargement of white adipocytes, formation of lipid droplets that accumulate excessive energy, and qualitative changes in the amount of secretion of various physiologically active substances.

Under such circumstances, various techniques have been proposed to eliminate or prevent obesity. For example, Patent Document 1 discloses a lipolysis promoter that is effective in improving obesity by promoting the reduction of whole-body or local adipose tissue, or suppressing or preventing obesity by preventing the increase in the same tissue. ing.

On the other hand, it has been found that, in addition to hypertrophy of adipocytes, an increase in the number of adipocytes due to differentiation from preadipocytes to adipocytes itself is a major factor in obesity formation. In fact, it has been pointed out that the number of white adipocytes increases after adulthood and is involved in the progression of obesity. Therefore, it is expected that suppressing the differentiation of pre-white adipocytes and suppressing the increase in the number of mature white adipocytes contributes to amelioration of obesity.

As a technology that induces an anti-obesity effect by suppressing the differentiation of adipocytes, for example, in Patent Document 2, an O-acyl derivative of the acidic mucopolysaccharide WAK-1-A produced by the marine bacterium Pseudomonas sp. A preadipocyte differentiation inhibitor is disclosed. Further, for example, in Patent Document 3, a fat accumulation inhibitor, an adipose precursor cell differentiation inhibitor, a visceral fat-reducing agent, and a visceral fat-reducing agent containing avian eggs containing 4.2 ppm by mass or more of iodine as an active ingredient Disclosed is a food and drink for In addition, Patent Document 4 discloses a preadipocyte differentiation inhibitor containing at least one selected from a group of mushrooms such as Versicolor versicolor and valerian, plants, and plant parts.

Here, melanin related to the present technology will be described. Melanin is a natural product produced in vivo by enzymatic oxidation from the amino acid tyrosine via L-Dopa and polymerization reaction. It is a black pigment produced in human hair and skin by ultraviolet irradiation, and is also the main pigment component of squid ink. Dopa-melanin is a black substance produced by polymerizing L-Dopa as an initial substance in an alkaline solution, and is considered an artificial melanin. The exact structure of either natural melanin or synthetic dopa-melanin has not been clarified, but a putative structure has been proposed.

As a technique using melanin, for example, Patent Document 5 discloses an antiallergic agent containing solubilized melanin or dispersed melanin as an active ingredient. In addition, Patent Document 6 discloses a cell growth inhibitor containing solubilized melanin or dispersed melanin as an active ingredient.

JP-A-2000-169325 JP 2007-77025 A WO 2018/051471 Pamphlet JP-A-2004-75640 JP 2018-2663 A JP 2018-20991 A

As described above, if the differentiation from preadipocytes to adipocytes can be suppressed, obesity can be prevented and the progression of obesity can be prevented. It is expected to contribute to prevention of exacerbation of habitual diseases and metabolic syndrome in which these diseases overlap.

Therefore, the main object of the present technology is to provide a technology for suppressing the differentiation of preadipocytes into adipocytes.

The inventors of the present application conducted intensive research on techniques for suppressing the differentiation of preadipocytes into adipocytes, and as a result, found that melanin has a preadipocyte differentiation inhibitory effect, leading to the completion of the present technology. .

That is, the present technology first provides a preadipocyte differentiation inhibitor containing melanin as an active ingredient.
The preadipocyte differentiation inhibitor according to the present technology may contain melanin and melanoprotein derived from squid ink.
In this case, enzyme-treated squid-derived melanin and/or enzyme-treated melanoprotein can be used.
The preadipocyte differentiation inhibitor according to the present technology may contain synthetic melanin.
The preadipocyte differentiation inhibitor according to the present technology may contain colloidal melanin and/or colloidal melanoprotein.

Next, the present technology provides food and drink for anti-obesity or anti-metabolic syndrome containing the preadipocyte differentiation inhibitor according to the present technology.
In addition, an anti-obesity or anti-metabolic syndrome drug containing the preadipocyte differentiation inhibitor according to the present technology is also provided.
Furthermore, the present technology provides an anti-obesity or anti-metabolic syndrome cosmetic containing the preadipocyte differentiation inhibitor according to the present technology.

The present technology further provides a method of preventing or treating obesity or metabolic syndrome (excluding medical practice for humans), which comprises administering melanin.

In addition, in this technique, "melanoprotein" is a technical term indicating a melanin-containing protein complex. Melanin is a natural pigment produced by various animals, plants, insects, fungi, and eubacteria. That is, it exists in the form of melanoprotein. That is, "melanoprotein" is a general term for proteins that bind to melanin. Melanoprotein amino acid residues include acidic amino acids, basic amino acids, and amino acids having hydroxyl groups and sulfhydryl groups (thiol groups) in their side chains. Melanoproteins naturally exist widely in the living world, for example, in squid ink and the hair of animals including humans. The protein composition and structure differs depending on the species. For example, compared to human hair-derived melanoprotein, melanoprotein derived from squid ink has much lower solubility in alkaline solutions and phosphate buffers, and is more strongly agglomerated. It is considered. Melanoproteins are spherical particles approximately 100 nm to 400 nm in diameter and exist as aggregates with varying numbers of associations.

Melanoidins (brown components of miso and soy sauce), which are dark brown substances produced by the reaction of amino acids and sugars called the Maillard reaction, are sometimes expressed as melanoproteins when combined with proteins. melanoprotein” is completely different. That is, "melanoprotein" in the present technology does not include melanoidin-containing proteins.

1 is a conceptual diagram showing the mechanism of adipocyte differentiation. FIG. It is a pie chart which shows the main component ratio of squid ink. Example (2) It is a conceptual diagram showing the differentiation induction schedule and the administration period pattern of synthetic melanin in the suppression of adipocyte differentiation by synthetic melanin. FIG. 4 is a graph showing oil droplet staining images of mouse preadipocytes 3T3-L1 at the end points of patterns a to c shown in FIG. 3 and the quantitative results thereof. FIG. FIG. 10 is a graph showing the quantitative results of mouse preadipocyte 3T3-L1 at the end point in the inhibition of adipocyte differentiation by squid ink in Example (3). Fig. 4 is a graph showing the expression results of differentiation-related genes in the expression analysis of adipocyte differentiation-related factors at the gene level in Example (4). Example (5) It is a band signal image of a differentiation-related protein in expression analysis at the protein level of adipocyte differentiation-related factor. Fig. 10 is a graph showing the results of expression of genes that respond to TNF-α stimulation in the study of the effect of melanin on genes that respond to TNF-α stimulation in Example (6). Example (7) Photographs and graphs showing the results of a macrophage migration test using a cell culture insert. Example (8) In the effect of melanin on obesity model due to high-fat diet load, FIG. These results show transitions.

A preferred embodiment for implementing the present technology will be described below. It should be noted that the embodiments described below are examples of representative embodiments of the present technology, and the scope of the present technology should not be construed narrowly.

1. Preadipocyte Differentiation Inhibitor FIG. 1 is a conceptual diagram showing the mechanism of adipocyte differentiation. Adipocytes are cells that develop from mesodermal pluripotent stem cells, pass through lipoblasts, and differentiate into preadipocytes, and various adipocytokines are secreted from adipocytes. For example, small adipocytes secrete beneficial cytokines such as adiponectin, which improve insulin sensitivity, promote fat burning, and prevent arteriosclerosis.

However, due to lack of exercise and excessive calorie intake, adipocytes gradually become hypertrophied, and when hypertrophic adipocytes occur, regulation of adipocytokine production is disrupted. Specifically, bad cytokines such as MCP-1 (monocyte chemoattractant protein-1), TNF-α (tumor necrosis factor alpha), IL-6 (interleukin-6), free fatty acid (FFA: Free Fatty Acid), etc. While the production of promoting factors increases, the production of anti-inflammatory factors such as the beneficial cytokine adiponectin decreases, resulting in increased inflammation.

The preadipocyte differentiation inhibitor according to the present technology has an action of suppressing the differentiation of preadipocytes into adipocytes. In addition, as shown in the examples below, it also has the effect of suppressing the decrease in the expression of adiponectin, which is a beneficial cytokine.

Patent Document 6 describes that solubilized melanin or dispersed melanin has an effect of suppressing the proliferation of cancer cells, but the proliferation of cancer cells and the differentiation of preadipocytes are completely different concepts. . That is, proliferation of cancer cells is the abnormal proliferation of undifferentiated cells, and the proliferation of cancer cells is stopped by promoting differentiation. Therefore, it is possible to suppress the growth of cancer cells by promoting differentiation. Conversely, inhibition of differentiation may promote proliferation of cancer cells.

In general, inhibition of preadipocyte differentiation is often accompanied by toxicity, and it is difficult to completely inhibit preadipocyte differentiation while ensuring cell safety. On the other hand, the preadipocyte differentiation inhibitor according to the present technology can inhibit almost 100% of the differentiation of preadipocytes without showing almost any cytotoxicity, as shown in the examples described later.

In addition, the preadipocyte differentiation inhibitor according to the present technology does not suppress enlargement of adipocytes, as shown in Examples described later. In other words, it does not inhibit the function of the amount of adipocytes required for a healthy body.

By suppressing the differentiation of adipocytes using the preadipocyte differentiation inhibitor according to the present technology, diseases or symptoms caused by an increase in adipocytes can be prevented or treated. Specifically, obesity, metabolic syndrome, and the like can be prevented or treated by using the preadipocyte differentiation inhibitor according to the present technology.

In the present technology, "prevention" of a disease or condition includes preventing the occurrence of the disease or condition, delaying the occurrence, and reducing the risk of occurrence. In addition, even when a disease or symptom has already occurred (affected, developed), suppression of further progress of the disease or symptom may also be included in "prevention". "Treatment" means alleviating, alleviating or slowing the rate of progression of a disease or symptom. Therefore, in the present technology, the technical meanings of "prevention" and "treatment" may overlap each other.

The preadipocyte differentiation inhibitor according to the present technology contains melanin as an active ingredient. The technical term “melanin” used in the present technology is used as a term encompassing various structurally similar melanins (eumelanin, pheomelanin, neuromelanin, allomelanin, phytomelanin, etc.). Melanin is a pigment found in various animals, plants, insects, fungi, eubacteria, and the like. The origin of melanin in the present technology is not particularly limited. Melanin is synthesized by chemical synthesis (see, for example, Japanese Patent Application Publication No. 2003-509529), enzymatic synthesis (see, for example, JP-A-07-313155), natural products (eg, squid ink, octopus ink, mushrooms, bananas, morning glory seeds, etc.). prepared by extraction and/or purification from melanocytes (mouse B16, B16F10, Syrian hamster RPMI 1846, human HMY-1, MNT-1, HM3KO, A375, SK-Mel-28, etc.) can do. In addition, various types of melanin are commercially available (for example, melanin oxidatively synthesized by treating tyrosine with hydrogen peroxide provided by Sigma-Aldrich, and melanin oxidatively synthesized by treating tyrosine with persulfate provided by MP-Biochemicals). melanin) and the like may also be used.

The purity or degree of refinement of melanin is also not particularly limited. Therefore, as long as it exhibits the expected effects, that is, preadipocyte differentiation inhibitory activity, for example, natural products containing melanin (for example, natural products such as squid ink as they are), crudely purified melanin derived from natural products, etc. It is also possible to use unpurified melanin or relatively low-purified melanin.

The form of melanin in the preadipocyte differentiation inhibitor according to the present technology is not particularly limited, and the above-described melanin-containing natural product itself, melanin alone, or a complex of melanin and other substances (e.g., proteins, etc.) bodies (eg, melanoproteins, etc.), and combinations thereof.

It is preferable to use one or more selected from squid ink-derived melanin, melanoprotein, and synthetic melanin for the preadipocyte differentiation inhibitor according to the present technology. A mixture of two or more types of melanin that differ in structure and/or molecular weight may be used as the active ingredient.

Synthetic melanins include eumelanin, namely 5,6-dihydroxyindole (DHI), indole-5,6-quinone (IQ), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), indole-5,6 -Quinone-2-carboxylic acid (IQCA) is preferably used as a monomer.

As the sac sac-derived melanin and/or melanoprotein, it is preferable to use sac sac-derived melanin and/or enzyme-treated melanoprotein treated with a digestive enzyme. As shown in Examples described later, by treating squid ink-derived melanin and/or melanoprotein with a digestive enzyme, the preadipocyte differentiation inhibitory effect can be further improved.

The type of digestive enzyme that processes squid ink-derived melanin and/or melanoprotein is not particularly limited. For example, proteolytic enzymes, lipid-degrading enzymes, carbohydrate-degrading enzymes, etc. can be mentioned, and in the present technology, one or more of these can be selected and used. In this technique, it is preferable to use a digestive enzyme having proteolytic ability. As shown in FIG. 2, about 75% of the components of squid ink are water, and when water is removed, melanin is about 62%, protein is about 25%, and lipid is about 12%. Among the components of squid ink, protein is the second most abundant component after melanin. Therefore, by decomposing protein, it is possible to further improve the preadipocyte differentiation inhibitory effect of melanin.

In addition, melanin and melanoprotein in the preadipocyte differentiation inhibitor according to the present technology are preferably present in a colloidal form. By existing in a colloidal form, the preadipocyte differentiation inhibitory effect can be further improved.

The preadipocyte differentiation inhibitor according to the present invention may contain one or more of various components according to the final product by freely selecting them, as long as the effect of the present technology is not impaired. For example, components such as preservatives, stabilizers, preservatives, emulsifiers, pH adjusters, thickeners, spices, seasonings, enzymes, vitamins, minerals, pigments and fragrances can be used.

2. Food and Beverage Products for Antiobesity or Antimetabolic Syndrome The preadipocyte differentiation inhibitor according to the present technology can be used in food and drink products for antiobesity or antimetabolic syndrome by utilizing its preadipocyte differentiation inhibitory action. The food and drink according to the present technology can be prepared by adding the preadipocyte differentiation inhibitor according to the present technology to a known food and drink, and the preadipocyte differentiation inhibitor according to the present technology can be added to the ingredients of the food and drink. can be mixed to produce a new food or drink.

Food and drink for anti-obesity or anti-metabolic syndrome according to this technology, regardless of the form such as liquid, paste, solid, powder, etc. In addition to tablet confectionery, liquid diet, etc., for example, flour products, instant foods, agricultural processing products, processed marine products, processed livestock products, milk/dairy products, oils and fats, basic seasonings, compound seasonings/foods, frozen foods, confectionery, beverages, and other commercially available products.

In addition, the anti-obesity or anti-metabolic syndrome food and drink according to the present technology can be provided and sold as food and drink labeled with specific uses (especially health uses) and functions. For example, provided as health food, functional food, food for the sick, enteral nutrition food, food for special dietary use, food with health claims, food for specified health use, food with function claims, food with nutrient function claims, quasi-drugs, etc. can be sold.

The anti-obesity or anti-metabolic syndrome food or drink according to the present technology contains an amount of melanin necessary for preventing or treating obesity or metabolic syndrome. The amount of melanin in the anti-obesity or anti-metabolic syndrome food and drink according to the present technology can be freely set as long as it does not impair the effect of the present technology, but is set in the range of 0.1 to 95% by weight. preferably.

Food and drink for anti-obesity or anti-metabolic syndrome according to the present technology include humans and non-human mammals (pet animals, domestic animals, experimental animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows , pigs, goats, sheep, dogs, cats, chickens, quails, etc.).

3. Anti-obesity or anti-metabolic syndrome drug The preadipocyte differentiation inhibitor according to the present technology can be used as an anti-obesity or anti-metabolic syndrome drug by utilizing its preadipocyte differentiation inhibitory action. The pharmaceutical according to the present technology can be prepared by adding the preadipocyte differentiation inhibitor according to the present technology to a known pharmaceutical, or by mixing the preadipocyte differentiation inhibitor according to the present technology into the raw material of the pharmaceutical. It is also possible to manufacture new pharmaceuticals using

Pharmaceutical preparations using the preadipocyte differentiation inhibitor according to the present technology can be formulated according to conventional methods. When formulating, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline solution, etc.). Lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used as excipients. Starch, carboxymethyl cellulose, calcium carbonate and the like can be used as the disintegrant. Phosphate, citrate, acetate and the like can be used as buffers. Gum arabic, sodium alginate, tragacanth and the like can be used as emulsifiers. As suspending agents, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used. Benzyl alcohol, chlorobutanol, sorbitol and the like can be used as analgesics. Propylene glycol, ascorbic acid and the like can be used as stabilizers. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. As antiseptics, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used.

In addition, the anti-obesity or anti-metabolic syndrome drug of the present technology may contain other active ingredients in addition to melanin. For example, by containing melanin and an active ingredient having a lipolytic action, obesity can be prevented by suppressing the differentiation of adipocytes and suppressing the increase in the number of adipocytes while promoting the decomposition of existing adipocytes. and can contribute to the prevention and treatment of metabolic syndrome. In addition, it is possible to prevent or treat metabolic syndrome from multiple perspectives by combining it with a preventive or therapeutic agent for diabetes, a preventive or therapeutic agent for hypertension, a preventive or therapeutic agent for hyperlipidemia, or the like.

The dosage form for formulation is also not particularly limited, and examples include nasal drops, eye drops, tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories. Can be formulated.

An anti-obesity or anti-metabolic syndrome pharmaceutical according to the present technology comprises an amount of melanin necessary for prevention or treatment of obesity or metabolic syndrome. The amount of melanin in the anti-obesity or anti-metabolic syndrome drug according to the present technology can be freely set as long as it does not impair the effect of the present technology, but is set in the range of 0.1 to 95% by weight. is preferred.

Anti-obesity or anti-metabolic syndrome pharmaceuticals according to the present technology may be administered orally or parenterally (intravenous, intraarterial, subcutaneous, intramuscular, or intraperitoneal injection, transdermal, nasal, transdermal) depending on the dosage form/form. mucous membranes, coatings, etc.). The "subject" here is not particularly limited, and includes humans and non-human animals (pet animals, domestic animals, experimental animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, chickens, quail, etc.).

The dose/use amount of the anti-obesity or anti-metabolic syndrome drug according to the present technology can be freely set as long as the effect of the present technology is not impaired. In setting an effective dosage, symptoms, age, sex, weight, etc. of the subject are generally taken into consideration. A person skilled in the art can set an appropriate dosage in consideration of these matters. As an administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. When creating an administration/usage schedule, it is possible to take into consideration the symptoms of the subject of application and the duration of the effect of the active ingredient.

4. Anti-obesity or anti-metabolic syndrome cosmetics The preadipocyte differentiation inhibitor according to the present technology can be used in anti-obesity or anti-metabolic syndrome cosmetics by utilizing its preadipocyte differentiation inhibitory action. The cosmetic according to the present technology can be prepared by adding the preadipocyte differentiation inhibitor according to the present technology to a known cosmetic, or the preadipocyte differentiation inhibitor according to the present technology can be added to the raw material of the cosmetic. can be mixed to produce a new cosmetic.

The anti-obesity or anti-metabolic syndrome cosmetic according to the present technology comprises the pre-adipocyte differentiation inhibitor according to the present technology and ingredients and base materials usually used in cosmetics (for example, various oils and fats, mineral oil, vaseline, Squalane, lanolin, beeswax, denatured alcohol, dextrin palmitate, glycerin, glycerin fatty acid ester, ethylene glycol, paraben, camphor, menthol, various vitamins, zinc oxide, titanium oxide, benzoic acid, edetic acid, chamomile oil, carrageenan, chitin powder , chitosan, perfume, coloring agent, etc.).

Embodiments of anti-obesity or anti-metabolic syndrome cosmetics according to the present technology are in the form of face or body emulsions, lotions, creams, lotions, essences, oils, packs, sheets, cleansers, and the like. can be done.

The anti-obesity or anti-metabolic syndrome cosmetic according to the present technology contains an amount of melanin necessary for preventing or treating obesity or metabolic syndrome. The amount of melanin in the anti-obesity or anti-metabolic syndrome cosmetic according to the present technology can be freely set as long as it does not impair the effects of the present technology, but is set in the range of 0.1 to 60% by weight. preferably.

5. Method for Preventing or Treating Obesity or Metabolic Syndrome Using the inhibitory effect of melanin on preadipocyte differentiation, the present technology provides a method for preventing or treating obesity or metabolic syndrome. A method for preventing or treating obesity or metabolic syndrome according to the present technology is a method including the step of administering melanin.

The method of administering melanin is not particularly limited as long as it does not impair the effects of the present technology. Specific examples of the administration method and the administration target are the same as the application method and the application target of the pharmaceutical product, and thus the description is omitted here.

The dose of melanin, administration schedule, etc. can be freely set as long as the effects of the present technology are not impaired. Specific examples of the dosage and administration schedule are also the same as the dosage/usage and administration schedule of the drug, and thus the description is omitted here.

Hereinafter, the present technology will be described in further detail based on examples. It should be noted that the embodiments described below are examples of representative embodiments of the present technology, and the scope of the present technology should not be interpreted narrowly.

(1) Materials and methods [Synthesis of melanin]
780 mL of ultrapure water was added to 2 g of L-Dopa (Sigma), 20 mL of 1N NaOH (FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was incubated with stirring at room temperature for 3 days while aeration was performed using an air pump. 0.2 mL of concentrated hydrochloric acid was added to 45 mL of the reaction solution, and the mixture was centrifuged at 3000 rpm for 5 minutes to obtain a precipitate. The supernatant was discarded, 40 mL of ultrapure water and 0.1 mL of concentrated hydrochloric acid were added to the precipitate, mixed, and centrifuged at 3000 rpm for 5 minutes. After repeating the same operation four times in total, the supernatant was discarded, 2 mL of 0.025N NaOH was added, all the samples were combined, and the pH was adjusted to 7.4 with a 1N NaOH solution. About 80 mL of this was placed in a dialysis tube (Spectrum CE dialysis tube, MWCO 20,000, area width 31 mm), and desalted and dialyzed in 10 L of deionized water for 2 days. The dialyzed sample was recovered, freeze-dried, and then stored frozen, or dissolved in ultrapure water or Hepes-KOH buffer (500 mM, pH 7.4) to an arbitrary concentration and stored at 4°C.

[Cells and culture]
Mouse preadipocyte 3T3-L1 cells (obtained from Human Science Research Resource Bank) were used as adipocytes in low-glucose DMEM medium supplemented with 10% fetal bovine serum, penicillin (100IU/mL), and streptomycin (100μg/mL). (FUJIFILM Wako Pure Chemical Corporation, 041-29775). Mouse RAW264 cells (obtained from RIKEN BRC) were used as macrophage cells in RPMI medium supplemented with 10% fetal bovine serum, penicillin (100IU/mL), and streptomycin (100μg/mL) (FUJIFILM Wako Pure Chemical Corporation, 189-02025). was subcultured in Culture was performed in an incubator (SANYO) at 5% CO ₂ and 37°C.

[Adipose cell differentiation induction method]
3T3-L1 cells that had become semi-confluent in a 10cm dish were detached with trypsin, the cells were seeded in a 24-well culture dish using high-glucose DMEM medium (FUJIFILM Wako Pure Chemical Corporation, 043-30085), and cultured for 3 days. After confirming that the cells became confluent, the cells were further cultured for one day. On the first day of induction of differentiation, the high-glucose DMEM medium was replaced with a differentiation-inducing medium supplemented with 1 μM dexamethasone, 250 μM 3-Isobutyl 1-methylxanthine, and 2 μM insulin, and cultured for 3 days. On day 3, the medium was replaced with a high-glucose DMEM medium supplemented with only 2 μM insulin, and thereafter, the medium was replaced with the same medium every two days and cultured.

[Oil Red O staining, quantification]
After washing the cells with PBS three times, they were fixed with a fixative (FUJIFILM Wako Pure Chemical Corporation, 10% neutral formaldehyde solution) at room temperature for 10 minutes. The fixative was removed, washed twice with PBS, treated with 60% isopropanol for 1 minute, and immediately after removal, Oil Red O (Sigma) staining solution was added. After treatment at room temperature for 20 minutes, the staining solution was removed, and the cells were washed once with 60% isopropanol and twice with PBS, and then observed under a bright field microscope. PBS was removed from the stained cells, isopropanol was added, and the cells were treated at room temperature for 1 hour. The oil red O solution was extracted and the absorbance at 490 nm was measured.

[Cell migration test (migration assay)]
RAW264 cells were cultured overnight in serum-free medium (starvation). A 24-well plate for migration assay was used, and 0.8 mL of 1% FBS-RPMI1640 medium diluted with an attractant (cytokine such as MCP-1 or TNF-α) was added to each well as necessary. Insert an insert (pore size 8 μm) there, add 0.3 mL of RAW264 cells suspended in 1% FBS-RPMI1640 medium to a concentration of 6.6×10 ⁵ /mL (2×10 ⁵ cells/well), and place in a CO ₂ incubator. and cultured. In addition, each concentration of melanin was added to the insert side as needed. After 24 hours, it was immersed in 4% paraformaldehyde phosphate buffer for 20 minutes and stained with 0.1% crystal violet solution for 60 minutes. After replacing with ultrapure water and allowing to stand for 1 hour, the cells inside the insert were gently scraped off with a cotton swab in order to detect only the cells that migrated to the outside of the insert due to migration. , a preparation was prepared and observed under a microscope.

[Western blotting]
After washing the cells with PBS three times, the cells were suspended in lysis buffer (30 mM Tris-HCl (pH 8.0), 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktails) and lysed by sonication. was disrupted, centrifuged at 20000×g at 4° C. for 30 minutes, and the supernatant was recovered. After quantifying the protein concentration of each sample by the BCA method using a commercially available kit (Pierce ^™ BCA Protein Assay Kit, Thermo Fisher Scientific), the protein concentration of all samples was adjusted to a constant value. A 4× sample buffer (FUJIFILM Wako Pure Chemical Corporation) containing 3-mercapto-1,2-propanediol was added to each sample, heat-treated at 100°C for 5 minutes, and stored at -30°C.
Sample proteins were separated by SDS-PAGE using a 5-20% gradient gel (SuperSep ^™ Ace, FUJIFILM Wako Pure Chemical Corporation). The amount of protein applied was 10-20 μg per lane. After SDS-PAGE electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore) using a semi-dry blotting apparatus (Horise Blot, ATTO). The membrane was blocked with 5% skimmed milk (PBS-T) (room temperature, 1 hour) and reacted overnight at 4°C with a specific antibody. After washing the membrane with PBS-T, it was treated with the corresponding secondary antibody at room temperature for 1 hour. After washing the membrane with PBS-T, it was treated with a chemiluminescent reagent (Western Lightning Plus-ECL, PerkinElmer), and band signals were detected using Light-Capture (ATTO).

[Gene expression quantification by real-time PCR method]
RNA was extracted from the cells using a commercially available RNA extraction kit (NucleoSpin® RNA, Macherey-Nagel). After the RNA concentration of each sample was quantified and adjusted to a constant concentration, cDNA was synthesized using a commercially available kit (ReverTra Ace (registered trademark), TOYOBO). Next, PCR reagents (THUNDERBIRD (registered trademark) SYBR qPCR Mix, TOYOBO) and gene-specific primers were mixed and subjected to a dedicated thermal cycler (Applied Biosystems (registered trademark) 7500 real-time PCR system, Thermo Fisher Scientific or CFX96 Touch Deep Well real-time PCR). Real-time PCR was performed using a PCR analysis system, BioRad). The primer sets used are shown in Table 1 below.

[Enzymatic treatment of squid ink]
Ikasumi powder (Nippon Chlorophyll Co., Ltd.) was used as the squid ink. Ikasumi powder was suspended in ultrapure water so as to be 2% (w/v). Peptidase R and protease A "Amano" SD manufactured by Amano Enzyme Co., Ltd. were used as enzymes. Alternatively, suspend in artificial intestinal fluid (50 mM, KH ₂ PO ₄ , pH 6.8), add the enzyme to an arbitrary concentration (0.1-1% (w/v)), and shake at 37°C for 3 days. Incubate while stirring. It was treated at 100°C for 10 minutes to inactivate the enzyme, sonicated at 30 second intervals for a total of 5 minutes, and then stored at 4°C.

[Animal experimentation]
Four-week-old male C57BL/6J mice (Japan SLC, Inc.) were obtained and acclimatized for two weeks. After measuring the body weight, 6 mice per group were confirmed by Bartlett test and One-way ANOVA to confirm that there was no significant difference in body weight between the groups. Experimental animals were reared in a light-dark controlled rearing room every 12 hours under a humidity of 50±10%, a temperature of 22±2°C, and an SPF environment. AIN-93M was used as a control diet, and HFD-60 (Oriental Bio Service Co., Ltd.) was used as a high-fat diet.

[Effect of melanin using high-fat diet model]
After acclimating 4-week-old mice for 2 weeks, normal diet control group (AIN93M + H ₂ O), normal diet melanin administration group (AIN93M + DM), high fat diet control group (HFD60 + H ₂ O), high They were divided into 4 groups of fat diet melanin administration group (HFD60+DM). Melanin was prepared at 100 mg/mL, and H ₂ O (0.2 mL) or melanin (100 mg/mL, 0.2 mL) was intragastrically administered every other day using a sonde (Shinano Seisakusho). The dose was set to 1,000 mg/kg as melanin amount. Mice were weighed every other day, the food was changed each time, and the amount fed and remaining was recorded. Cage exchange was performed once a week. Recordings were made for 12 weeks (84 days) (n=5/group).

(2) Suppression of adipocyte differentiation by synthetic melanin [differentiation induction schedule and administration period of synthetic melanin]
FIG. 3 shows the differentiation induction schedule and the pattern of administration period of synthetic melanin. First, in order to investigate whether melanin suppresses differentiation induction of adipocytes, a pattern in which melanin is added at the same time as differentiation induction stimulation was investigated (pattern a). Specifically, in pattern a, synthetic melanin was administered simultaneously with a differentiation inducer for 3 days, and then cultured for 7 days.

Next, to investigate whether melanin can contribute to the suppression of hypertrophy, synthetic melanin was administered after preadipocyte differentiation into small adipocytes, and the formation of intracellular oil droplets was evaluated 28 days after initiation of differentiation induction. (patterns b and c). Specifically, in pattern b, synthetic melanin was administered for 3 days starting 7 days after differentiation-inducing stimulation, after which cells were cultured in normal insulin-supplemented medium for up to 28 days. In pattern c, synthetic melanin was administered from day 7 after differentiation-inducing stimulation, and cultured until day 28.

In all of patterns a to c, after 3 days from the initiation of differentiation induction, only insulin was added to the medium, and the culture was continued until the end point while exchanging the medium every 3 days. Synthetic melanin was administered at 0.062, 0.125 and 0.25 mg/mL.

[Oil droplet staining image and its quantification]
At each endpoint of patterns a to c, the absorbance was measured after extracting Oil Red O dye using the method described in (1) Materials and Methods [Oil Red O staining, quantification]. The relative absorbance was shown by taking the absorbance of the sample with only differentiation induction as 100.

[result]
FIG. 4 shows oil droplet staining images of mouse preadipocytes 3T3-L1 at the endpoints of patterns a to c and their quantification results. As shown in Fig. 4a, when synthetic melanin was added for 3 days together with a 3-type mixed differentiation-inducing agent, the formation of intracellular oil droplets was suppressed in a melanin treatment concentration-dependent manner on day 7 after the initiation of differentiation induction. .

As shown in b and c of FIG. 4, when synthetic melanin was administered for 3 days on day 7 from the start of differentiation induction, and when synthetic melanin was continued to be administered from day 7 to day 28 of differentiation induction, in either case Also, no change in the amount of oil droplets was observed on the 28th day. That is, hypertrophy of adipocytes was not suppressed.

These results indicate that melanin does not suppress hypertrophy of differentiated adipocytes, but suppresses induction of differentiation.

(3) Suppression of adipocyte differentiation by squid ink Next, it was investigated whether squid ink and squid ink treated with an enzyme for food processing can suppress differentiation of adipocytes.

[Differentiation induction schedule and administration period of squid ink and enzyme-treated squid ink for food processing]
As in pattern a, squid ink and enzyme-treated squid ink for food processing are administered at the same time as the addition of the 3-kind mixed differentiation inducer, cultured for 3 days, then replaced with an insulin-added medium, and the medium is replaced every 2 days. and cultured for 7 days.

[Oil droplet staining image and its quantification]
On day 7 of the culture, using the method described in (1) Materials and Methods [Oil Red O staining, quantification], after Oil Red O staining, the dye was extracted and the absorbance at 490 nm was measured. The relative absorbance was shown with the absorbance of the sample that was only induced to differentiate as 100.

[result]
FIG. 5 shows the quantification results of mouse preadipocyte 3T3-L1 at the endpoint. As shown in FIG. 5A, squid ink was significantly inhibited by 84% against the positive control (PC) even without enzymatic treatment. In addition, when squid ink treated with two representative digestive enzymes for food processing at a concentration of 0.1% or 1% by weight, respectively, was administered, the differentiation induction inhibitory effect was significantly higher than when squid ink itself was administered. Admitted. There was no significant difference between 0.1% and 1% weight percent concentration of the enzyme.

FIG. 5B shows the results when untreated squid ink and enzyme-treated squid ink were co-administered with a differentiation-inducing agent at squid ink concentrations of 0.4, 0.8, and 1.6 mg/mL, respectively. Without enzyme treatment, differentiation was significantly suppressed in a dose-dependent manner (Dunnett test), and enzyme-treated squid ink inhibited differentiation more strongly than non-enzyme-treated squid ink at the same concentration (Student t-test). .

From the above results, it was shown that squid ink significantly inhibits adipocyte differentiation even without enzymatic treatment, but that enzymatic treatment inhibits differentiation more strongly.

Although the mechanism by which the enzymatic treatment improves the differentiation inhibitory effect is not clear, the melanoprotein contained in squid ink is decomposed by the enzymatic treatment, and the melanin contained in the melanoprotein is exposed, or the melanoprotein is released. Melanin and/or melanoprotein are incorporated into adipocytes by changes in steric structure (decomposition/deformation, etc.) or formation of aggregates of melanoproteins with fewer particles. It was presumed that it may contribute to the suppression of adipocyte differentiation by making it easier to bind to .

(4) Gene-level expression analysis of adipocyte differentiation-related factors After administration for 3 days, differentiation-related gene expression was examined by quantitative PCR using the method described in (1) Materials and Methods [Gene expression quantification by real-time PCR]. Rsp16 (40S ribosomal protein S16) was used as an endogenous control gene.

The results are shown in FIG. As shown in Figure 6, FABP4 (fatty acid binding protein 4), which plays a regulatory role in lipid droplet formation, transcription factor C/EBP α (CCAAT/enhancer-binding protein alpha), which controls adipocyte differentiation, and fatty acids. Enzyme protein Fasn (Fatty acid synthase), which plays a role in synthesis, Acox1 (acyl-CoA oxidase 1), which encodes the rate-limiting enzyme for β-oxidation of fatty acids, is mainly secreted by adipocytes and is involved in the regulation of glucose metabolism and fatty acid oxidation. The induction of expression of all adiponectins was suppressed in a melanin concentration-dependent manner.

(5) Expression analysis of adipocyte differentiation-related factors at protein level ) for 3 days, expression analysis of differentiation-related proteins was performed using the method described in [Western blotting] in (1) Materials and methods above. β-actin was used as a loading control.

Results are shown in FIG. The expression level of ACC (acetyl-CoA carboxylase), which is involved in the regulation of fatty acid synthesis and fatty acid β-oxidation, decreased in a melanin treatment concentration-dependent manner. Phosphorylation of Ser79, an ACC inactivation marker, was correlated with ACC protein levels, suggesting that melanin does not suppress the activation of the protein, but suppresses the protein expression itself. was suggested. In addition, PPARγ (The peroxisome proliferator-activated receptors gamma) and C/EBPα are called master regulators involved in the differentiation of progenitor cells into adipocytes, and act as transcription factors for protein expression characteristic of adipocytes. Melanin suppressed the protein expression levels of these master regulators in a dose-dependent manner. * indicates a non-specific band signal.

These results suggest that melanin suppresses adipocyte differentiation and oil droplet formation, that is, adipogenesis, by suppressing the expression of differentiation-related genes rather than by suppressing the activation of fatty acid synthesis proteins. strongly suggested.

(6) Investigation of Effect of Melanin on Genes Responding to TNF-α Stimulation The effect of melanin on differentiated adipocytes was examined. White adipocytes secrete various beneficial physiologically active substances, one of which is adiponectin. However, with obesity, its expression is suppressed by TNF-α produced by itself and by macrophages. Released into the stream, it is said to contribute to the onset and exacerbation of arteriosclerosis.

FIG. 8A shows the results of treating differentiated adipocytes with TNF-α and examining the expression of adiponectin. When differentiated adipocytes were treated with TNF-α, the adiponectin gene was repressed in a TNF-α dose-dependent manner.

FIG. 8B shows the results of treating differentiated adipocytes with TNF-α and examining the expression of the MCP-1 gene. When differentiated adipocytes were treated with TNF-α, MCP-1 gene expression was increased (Dunnett test).

FIG. 8C shows the results of pre-administering 0.125 mg/mL of melanin to differentiated adipocytes, treating the differentiated adipocytes with TNF-α, and examining the expression of adiponectin. Pretreatment with melanin inhibited the TNF-α-induced reduction of adiponectin gene expression. As mentioned above, since melanin treatment alone increased adiponectin gene expression, it is presumed that the suppression of the apparent decrease in adiponectin expression is the result of the increase in adiponectin expression due to melanin exceeding the decrease in gene expression due to TNF-α. rice field.

FIG. 8D shows the results of pre-administration of 0.5 mg/mL of melanin to differentiated adipocytes, treatment of TNF-α, and examination of MCP-1 gene expression. Melanin did not suppress the upregulation of MCP-1 by TNF-α.

From the above, it is possible that melanin does not suppress the expression of the inflammation-inducing chemokine MCP-1, although the decrease in adiponectin expression induced by TNF-α is consequently suppressed in differentiated adipocytes that are not hypertrophied. was done.

(7) Macrophage migration test using cell culture insert After administering the amount of melanin (0.0312, 0.0625mg/mL) shown in FIG. Using the method described in [Cell Migration Test (Migration Assay)], the effect of melanin on macrophage migration was confirmed.

FIG. 9A shows the results of measuring migratory cells by culturing a certain number of macrophages with each concentration of melanin using a culture supernatant (conditioned medium) obtained by treating differentiated adipocytes with TNF-α as an attractant. In the absence of melanin treatment, a large number of macrophages migrated, whereas in melanin-treated cells, the migration was inhibited in a dose-dependent manner.

FIG. 9B shows the result of adding only recombinant MCP-1 as an attractant to the medium and examining macrophage cells migrating in response. Macrophage migration was strongly inhibited in a melanin treatment concentration-dependent manner.

These results indicate that melanin suppresses macrophage migration due to adipocyte-derived chemotactic properties produced in response to TNF-α, including MCP-1. 8 and 9, melanin restores the expression of adiponectin, whose production is reduced by TNF-α, in differentiated white adipocytes, but cannot suppress the expression of MCP-1, which attracts macrophages. It was suggested that the prolongation of inflammation could be suppressed by suppressing the MCP-1 response.

(8) Effect of melanin on obesity model with high-fat diet load Using the method described in (1) Materials and methods [Animal experiment] and [Effect of melanin using a high-fat diet model], a high-fat diet We investigated the effects of melanin administration on obesity model mice caused by stress.

FIG. 10A shows the results showing changes in food intake in each group during the experimental period. No significant difference in food intake was observed between the groups.

FIG. 10B shows the results showing changes in body weight in each group during the experimental period. No significant difference was observed between the water-administered group and the melanin-administered group between the normal diet groups. On the other hand, in the high-fat diet-loaded group, the melanin-administered group showed a significant weight-suppressing effect after 7 weeks (49 days) of administration (Student t-test).

Claims (9)

A preadipocyte differentiation inhibitor containing melanin as an active ingredient. 2. The preadipocyte differentiation inhibitor according to claim 1, which contains melanin and melanoprotein derived from squid ink. 3. The preadipocyte differentiation inhibitor according to claim 2, which contains enzyme-treated squid ink-derived melanin and/or enzyme-treated melanoprotein. The preadipocyte differentiation inhibitor according to any one of claims 1 to 3, which contains synthetic melanin. 5. The preadipocyte differentiation inhibitor according to any one of claims 1 to 4, which contains colloidal melanin and/or colloidal melanoprotein. Food and drink for anti-obesity or anti-metabolic syndrome, containing the preadipocyte differentiation inhibitor according to any one of claims 1 to 5. An anti-obesity or anti-metabolic syndrome pharmaceutical containing the preadipocyte differentiation inhibitor according to any one of claims 1 to 5. An anti-obesity or anti-metabolic syndrome cosmetic containing the preadipocyte differentiation inhibitor according to any one of claims 1 to 5. A method for preventing or treating obesity or metabolic syndrome (excluding medical practice for humans), which comprises the step of administering melanin.

Images