JP7185990B2 - Adiponectin secretion promoter, adipocyte differentiation promoter, and pharmaceutical composition, food and feed containing them - Google Patents

Description

TECHNICAL FIELD The present invention relates to novel adiponectin secretagogues, adipocyte differentiation promoters, and pharmaceutical compositions, foods, and feeds containing them.

Obesity is a condition in which fat accumulates excessively in adipose tissue. In addition to genetic factors, environmental factors, especially lifestyle habits such as overeating and lack of exercise, are major factors in the development of obesity. . Metabolic syndrome (visceral fat syndrome) is defined as “a multiple risk factor syndrome that combines accumulation of visceral fat, insulin resistance based on it, abnormal glucose metabolism, abnormal lipid metabolism, and hypertension, leading to arteriosclerosis. It is said that it is in an easy state. The number of people with metabolic syndrome is estimated to be about 9.4 million and the number of potential patients is about 10.2 million, totaling about 19.6 million (survey in 2006).

Mammals including humans are known to have two types of adipose tissue. Of these, brown adipose tissue is an organ that decomposes fat and produces heat, whereas white adipose tissue exists as subcutaneous fat or visceral fat (intestinal fat). Recent studies have revealed that white adipose tissue is not just a fat storage site, but an endocrine tissue that produces and secretes various physiologically active substances and exerts a great influence on the body.

In addition, recent research results have drawn attention to the relationship between obesity and inflammation in adipose tissue. Obesity is a chronic inflammatory condition of adipose tissue that leads to abnormalities in adipocytokines, resulting in metabolic syndrome. Therefore, in addition to suppressing the most upstream obesity (visceral fat accumulation), normalization of adipose tissue inflammation and abnormal expression and secretion of adipocytokines due to obesity maintains adipose tissue and treats and prevents metabolic syndrome. It is considered one of the important targets.

In addition, recent studies have revealed that fat cells have different properties depending on their size. Small adipocytes in the viscera secrete the secretory protein adiponectin, but as adipocytes become hypertrophied, the amount of adiponectin secreted decreases and insulin resistance is induced. Furthermore, it is also known that hypertrophied adipocytes are reduced by apoptosis, and at that time, they release large amounts of insulin resistance-inducing substances such as TNF-α. In addition, insulin resistance refers to a state in which the sensitivity to insulin is reduced and the action of insulin cannot be sufficiently exerted. As a result of induction of insulin resistance, the glucose uptake capacity of muscle and adipose tissue is reduced, and gluconeogenesis cannot be suppressed in the liver. As a result, blood sugar levels are less likely to fall and more insulin is required to bring blood sugar levels back to normal. If this state continues, the insulin secretion function of the pancreas will decrease and the blood sugar level will increase, which is said to cause type II diabetes.

Since adiponectin is a recently identified protein, there are many unknowns about its action, etc. However, it promotes glucose uptake into cells not via insulin receptors and activates AMP kinase in liver and skeletal muscle. It has been reported that it reduces intracellular fatty acids and enhances insulin resistance. Thiazolidinedione derivatives such as pioglitazone are known as drugs that reduce hypertrophied adipocytes, increase small adipocytes, and promote the production of adiponectin (see, for example, Non-Patent Document 1).

In addition, a food-derived component that has an effect of promoting the production and/or suppressing the decrease of adiponectin is also being searched (see, for example, Patent Document 1).

JP 2015-61872 A

Hoshi University Open Research Center Website “Drugs for Diabetes” (URL: http://polaris.hoshi.ac.jp/openresearch/diabetes%20drugs.html) (“4. Insulin sensitizers, thiazolidinediones derivatives”)

However, drugs such as artificial compounds have side effects. On the other hand, some food-derived ingredients have low adiponectin secretion-promoting activity, and some do not exhibit differentiation-promoting activity of preadipocytes into small adipocytes.

The present invention has been made in view of such circumstances, and aims to provide a safe and highly active adiponectin secretagogue, an adipose precursor cell differentiation promoter, and a pharmaceutical composition, food, and feed containing one or both of these. aim.

A first aspect of the present invention in line with the above objects is to solve the above problems by providing an adiponectin secretagogue characterized by containing an extract of pericarp of Trapa bispinosa as an active ingredient. .

A second aspect of the present invention is to solve the above problems by providing an adipose precursor cell differentiation promoting agent comprising an extract of the pericarp of Trapa bispinosa as an active ingredient.

A third aspect of the present invention is to solve the above problems by providing a pharmaceutical composition for promoting adiponectin secretion, containing the adiponectin secretagogue according to the first aspect of the present invention.
A fourth aspect of the present invention is to solve the above problems by providing a pharmaceutical composition for promoting preadipocyte differentiation comprising the preadipocyte differentiation promoting agent according to the second aspect of the present invention.

A fifth aspect of the present invention solves the above problems by providing an adiponectin secretagogue- promoting food containing the adiponectin secretagogue according to the first aspect of the present invention.
A sixth aspect of the present invention solves the above problems by providing a food product for promoting preadipocyte differentiation containing the preadipocyte differentiation promoting agent according to the second aspect of the present invention.

A seventh aspect of the present invention solves the above problems by providing an adiponectin secretion promoting feed containing the adiponectin secretagogue according to the first aspect of the present invention.
An eighth aspect of the present invention solves the above problems by providing a preadipocyte differentiation promoting feed containing the preadipocyte differentiation promoting agent according to the second aspect of the present invention.

The compound, which is the active ingredient of the adiponectin secretion-promoting agent and the adipocyte differentiation-promoting agent according to the present invention, is contained in a plant belonging to the Uraraceae family that has been eaten, and its safety has been confirmed. In addition, as found in the present invention, it has high adiponectin secretion-promoting activity and adipose precursor cell differentiation-promoting activity. Thus, according to the present invention, safe and highly active adiponectin secretagogues and adipose precursor cell differentiation promoters are provided.

Fig. 10 is a graph showing the results of Oil Red O staining in a preadipocyte differentiation promotion test using a gallbladder extract. Fig. 10 is a graph showing the results of the amount of adiponectin produced in a differentiation promotion test of preadipocyte cells using a gallbladder extract. 1 is a graph showing changes in blood adiponectin levels in a human oral administration test using an extract from Galpin. 1 is a graph showing changes in blood adiponectin levels in female subjects in a human oral administration test using an extract of Marcelia japonicum.

The adiponectin secretion-promoting agent according to the first embodiment of the present invention and the preadipocyte differentiation-promoting agent according to the second embodiment of the present invention (hereinafter referred to as "adiponectin secretion-promoting agent" and "adipocyte differentiation-promoting agent", respectively) may be abbreviated as "agent".) contains as an active ingredient one or more compounds contained in one or both of the pericarp and fruit of a plant belonging to the Arboraceae family, each of which contains small adipocytes (white adipocytes ) and the activity of inducing or promoting the differentiation of preadipocytes into small adipocytes.

The one or more compounds contained in one or both of the pericarp and fruit of a lavender plant, which are the active ingredients of the adiponectin secretagogue and the adipose precursor cell differentiation promoter, include, for example, in the case of a composition for oral administration, , It may contain pulverized or finely powdered peels and fruits, but for example, in the case of manufacturing compositions used as injections, juice of fruits, one or both of fruits and peels is preferably used.

The one or more compounds contained in one or both of the pericarp and fruit of the Arboraceae plant are preferably an extract of one or both of the pericarp and fruit or one or more compounds separated from the extract. Methods for preparing one or both extracts of pericarp and fruit are described in detail below.

The fruits and pericarp of the arboraceous plant may be fresh fruits or collected from fresh fruits, dried fruits after collection, dried fruits or dried fruits collected from dried fruits. In order to improve extraction efficiency, pretreatment such as crushing or pulverization may be performed using any method prior to solvent extraction.

There are no particular restrictions on the tarpaulin family plant used for extraction, but specific examples include water chestnut (Trapa japonica), japonica (Trapanatans L. ver. japonica), himebishi (Trapa incisa), and horsetail (Trapa bispinosa Roxb.). be done.

As the solvent used for extraction, water, an aqueous solution, or a mixed solvent (aqueous solvent) obtained by mixing any one or more solvents miscible with water and water at any ratio can be used. Water, methanol, ethanol, and an aqueous solvent obtained by mixing any two or more of these at any ratio, and particularly preferred extraction solvents are water and ethanol, which is an organic solvent recognized as a food additive, and water. is an aqueous solvent mixed in a ratio of The temperature of the extraction solvent may be any temperature above room temperature and below the boiling point of the extraction solvent, but is preferably determined in consideration of extraction efficiency, heat resistance, volatility, and the like of the material to be extracted.

When water or an aqueous solvent is used as an extraction solvent, an acid, a base, a salt, or the like may be included as appropriate in order to improve the extraction efficiency. The temperature and pH of the water used for extraction are not particularly limited, but the pH is preferably near neutral, more specifically pH 4 to 9, and 6 to 8, in consideration of use in living organisms. It is more preferable to have If desired, heated extraction solvent may be used to improve extraction efficiency.

Solvent extraction can be carried out by any known method. For example, after mixing one or both of the pericarp and fruit of a lavender plant in a solvent for a predetermined period of time, filtration, centrifugation, decantation, or the like is used to separate the solid content. A method such as a continuous extraction method such as a Soxhlet extraction method can be used.

Prior to separating one or more compounds from the solvent extract of the pericarp or fruit of the lavender plant, dialysis, ultrafiltration, filtration, column chromatography or the like is used to remove high molecular weight components, insolubles, etc. Pretreatment may be performed.

When insoluble matter is removed by filtration, an adsorbent such as activated carbon, bentonite, or celite, or a filter aid may be added as necessary to remove impurities. In particular, when it is used in the form of an extract, it is preferable to carry out sterilization filtration with a membrane filter or the like.

Separation of the solvent extract pretreated as described above can be performed using any known method such as column chromatography, reversed-phase chromatography, ion chromatography, etc., which will be described later. It can be carried out by fractionating fractions having various high activities thought to be related to promotion of adiponectin secretion and induction or promotion of differentiation of pre-adipocytes into small adipocytes.

The evaluation of the activity of promoting the secretion of adiponectin of a solvent extract or a fraction thereof of pericarp or fruit of a narcissus plant (hereinafter sometimes abbreviated as "extract etc.") can be carried out either in vitro or in vivo. can also be performed, and in a culture test of small adipocytes or adipose precursor cells on a medium containing an extract, etc., or an administration test for humans or animals, the secretion amount of adiponectin as an indicator of the amount of adiponectin produced Alternatively, it can be performed by quantifying the blood adiponectin concentration. Adiponectin can be quantified using any known method such as the ELISA method.

To evaluate the activity of extracts, etc. to induce or promote the differentiation of preadipocytes into adipocytes, perform a culture test of preadipocytes on a medium containing extracts, etc., and quantify the amount of fat accumulated after culturing. It can be done by Quantification of the amount of accumulated fat is carried out using any known method, such as comparing the absorbance before and after culture using a fat-soluble dye such as Oil Red O, which selectively or specifically stains neutral fat. It can be carried out.

The adiponectin secretagogue and the adipocyte differentiation-promoting agent may be the same extract, fraction or isolated compound obtained by the above method, or different extracts, fractions or isolated It may be a compound.

By mixing an adiponectin secretion-promoting agent or an adipose precursor cell differentiation-promoting agent with a carrier or the like, it can be used as a pharmaceutical composition having one or both of therapeutic and preventive effects on diabetes and diseases and symptoms associated therewith. The dosage form of the pharmaceutical composition for humans or animals includes oral, rectal, parenteral administration (eg, intravenous administration, intramuscular administration, subcutaneous administration, etc.). , method of administration, purpose of use, and the age, body weight, and symptoms of the subject to which it is applied, and it is difficult to determine univocally. , preferably 0.1 to 2000 mg/day for adults. Of course, since the dose varies depending on various conditions, there are cases where a dose less than the above dose is sufficient, or there are cases where a dose exceeding the above range is necessary.

When it is prepared as an oral preparation, it can be prepared in the form of tablets, granules, powders, capsules, coating agents, liquid preparations, suspensions, etc. When it is prepared as a parenteral preparation, it can be prepared as an injection, a drip, It can be prepared in the form of a suppository and the like. Any known method can be used for formulation. For example, an adiponectin secretagogue or an adipose precursor cell differentiation promoter, a pharmaceutically acceptable carrier or diluent, a stabilizer, and other desired additives are blended to form the above desired dosage form. be able to.

Foods containing an adiponectin secretion-promoting agent or an adipose progenitor cell differentiation-promoting agent include those in which an adiponectin secretion-promoting agent or an adipose progenitor cell differentiation-promoting agent is blended with food, or capsules, tablets, etc., which are commonly used in foods or supplements. can take the form of There are no particular restrictions on the type of food to be blended, for example, liquid (fluid) food such as coffee, fruit juice, soft drink, beer, milk, miso soup, soup, black tea, tea, nutritional supplement, syrup, margarine, jam, etc. , cooked rice, bread, potato products, mochi, candy, chocolate, furikake, ham, sausage, solid foods such as candies, staple foods, side dishes, confectionery, and seasonings. It may be molded into a powder, granule, tablet, or the like, depending on the application. In addition, if necessary, excipients, bulking agents, binders, thickeners, emulsifiers, coloring agents, flavoring agents, food additives, seasonings and the like may be appropriately mixed.

In addition to foods and supplements for human consumption, when adiponectin secretagogues or adipose precursor cell differentiation promoters are mixed in feed and administered to mammals such as livestock and pets, the feed must be prepared in advance. It can be mixed into raw materials to prepare a functional feed. Moreover, an adiponectin secretion promoter or an adipose precursor cell differentiation promoter can be added to feed for administration. That is, a feed containing an adiponectin secretagogue or an adipose precursor cell differentiation promoter as an active ingredient can be safely added to livestock such as pigs, cattle, horses and sheep, and pets (dogs and cats). , can be used as a functional feed having an activity to improve diseases such as diabetes in these animals.

The present invention provides an adiponectin secretagogue or adipose precursor cell differentiation promoter containing, as an active ingredient, one or more compounds contained in one or both of the pericarp and fruit of a narcissus plant, thereby treating human diabetes and the like. Methods of ameliorating diseases and methods of ameliorating diseases such as diabetes in mammals other than humans are also provided.

Next, an example conducted to confirm the effects of the present invention will be described.
Example 1: Preparation of water chestnut peel extract and preparation for oral administration containing the same (1) Preparation of hot water extract of water chestnut (hereinafter abbreviated as "water chestnut peel extract") bispinosa) fruits were dried and the dried pericarp collected. Powder the dried pericarp using a food processor, hot water extraction (use 6 parts by weight of hot water at 90 ° C per 1 part by weight of dry pericarp), polyphenol content of the finally obtained water chestnut pericarp extract The extract was concentrated at a predetermined concentration ratio so that the content of the extract was 25% by weight or more. 33% by weight of dextrin was added to 67% by weight of the concentrate and spray-dried with a spray dryer. The powder thus obtained (with a polyphenol content of 25% by weight or more) was used as a water chestnut peel extract in the following tests.

(2) Preparation of Orally Administered Formulation Containing Water Chestnut Pericarp Extract To 100 mg of the water chestnut skin extract prepared in (1) above, 187 mg of cornstarch and 3 mg of calcium stearate were added as excipients, and the mixture was encapsulated in a gelatin hard capsule. In Example 3, which will be described later, the subjects were allowed to ingest the above hard capsules once a day, as described later. In addition, a placebo prepared using dextrin instead of the water chestnut peel extract was used as a placebo (indistinguishable from the orally administered drug containing the water chestnut peel extract).

Example 2: Test method for differentiation-inducing activity of adipose precursor cells:
Mouse-derived preadipocyte 3T3-L1 (DS Pharma Biomedical Co., Ltd.) was seeded on a 24-well collagen-coated plate (2×10 ⁴ cells/cm ² ) and cultured in DMEM basal medium (10% FBS, containing antibiotics). After culturing for 3 days, the cells were cultured in a differentiation-inducing medium (DMEM basal medium supplemented with 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/mL insulin) for 3 days. Next, after 4 days of culture in a differentiation-promoting medium (DMEM basal medium supplemented with 5 μg/mL insulin), Oil Red O staining and adiponectin concentration in the culture supernatant were measured. In addition, the pepper peel extract as a test substance and pioglitazone (Tokyo Chemical Industry Co., Ltd.) as a positive control were added to both the differentiation-inducing medium and the differentiation-promoting medium. Cells induced and promoted to differentiate without addition of the test substance were used as controls.

The Oil Red O staining method is one of the techniques for evaluating differentiation from preadipocytes to adipocytes. The method is shown below. After removing the cell culture supernatant from each well of the 24-well collagen-coated plate and washing twice with PBS, 0.2 mL of a 10% neutral buffered formalin solution (Wako Pure Chemical Industries) was added and allowed to stand at room temperature for 60 minutes. (Cell fixation) After that, the 10% neutral buffered formalin solution was removed, and the cells were washed twice with PBS. Then, 0.2 mL of 60% isopropyl alcohol was added, left at room temperature for 5 minutes and removed, and then Oil Red O staining solution ([Oil Red O]/[60% isopropyl alcohol] = 1.8 mg/mL, filtered through a filter). 0.2 mL was added and allowed to stand at room temperature for 15 minutes. After removing the Oil Red O staining solution, the plate was washed with distilled water three times, added with 0.2 mL of 100% isopropyl alcohol, and left at room temperature for 15 minutes or more to extract the pigment. 100 μL of the extract was transferred to a 96-well plate and the absorbance at 490 nm was measured using a microplate reader (TECAN). The adiponectin concentration contained in the culture supernatant was evaluated using a mouse/rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.). The results of measurement of absorbance and adiponectin concentration by Oil Red O staining were shown as relative values, with the control value being 1.

result:
<Differentiation promoting action to adipocytes>
As shown in FIG. 1, the results of absorbance measurement by Oil Red O staining confirmed a concentration-dependent increase in absorbance due to the pepper peel extract. In particular, at added concentrations of 5 and 10 μg/mL, it showed a significant increase in absorbance relative to the control, showing a higher value than the drug pioglitazone (0.25 μM). From the above results, it was shown that the pepper peel extract remarkably promotes the differentiation of preadipocytes into adipocytes.

<Adiponectin production promoting action in adipocytes>
As shown in FIG. 2, the measurement results of the adiponectin concentration in the culture supernatant confirmed a concentration-dependent increase in adiponectin due to the pepper peel extract, and a significant increase in the adiponectin concentration compared to the control at all concentrations. It has been shown. In particular, at an added concentration of 10 µg/mL, values close to those obtained when the drug pioglitazone (0.25 µM) was added were shown. These results indicated that the pepper peel extract strongly promoted adiponectin production from adipocytes.

Example 3: Test method for oral administration to human subjects:
Human studies were conducted in healthy males and females in a placebo-controlled, double-blind manner. The test period was 12 weeks. During the test period, the placebo intake group (12 people) received a placebo capsule, and the pepper peel extract intake group (13 people) received a capsule containing the pepper peel extract (approximately 66 mg/capsule in terms of the pepper peel extract), one capsule each day. ingested continuously. In addition, the subjects' blood was collected four times in total before ingestion, 4 weeks after ingestion, 8 weeks after ingestion, and 12 weeks after ingestion.

The concentration of adiponectin contained in the collected blood (plasma) was measured using a human adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.).

result:
The measurement results of the blood adiponectin concentration are shown in Tables 1, 2, 3 and 4. As shown in FIG. 3, the adiponectin concentration in the colonus pericarp extract intake group (water chestnut intake group) significantly increased after 12 weeks of intake compared to before intake (10.8→13.5 μg/mL). In addition, the amount of change in the adiponectin concentration before ingestion showed a significant increase in the water chestnut ingestion group compared to the placebo group at 12 weeks.

In addition, Table 2 and FIG. 4 show the results of extracting only female subjects and performing subclass analysis (6 females in the placebo group, 7 females in the water chestnut intake group). At week 12, the water chestnut-fed group showed a significant increase in adiponectin levels relative to the pre-fed and placebo groups.

From the above results, it was shown that continuous ingestion of the pepper peel extract increased the adiponectin concentration in the blood. As shown above, it was confirmed that the pepper peel extract exhibits the effect of significantly increasing adiponectin not only in vitro but also in vivo.

Claims (8)

An adiponectin secretagogue characterized by containing an extract of the pericarp of Trapa bispinosa as an active ingredient. An adipose precursor cell differentiation promoting agent comprising an extract of pericarp of Trapa bispinosa as an active ingredient. A pharmaceutical composition for promoting adiponectin secretion, comprising the adiponectin secretagogue according to claim 1. A pharmaceutical composition for promoting preadipocyte differentiation, comprising the preadipocyte differentiation promoting agent according to claim 2 . A food for promoting adiponectin secretion, comprising the adiponectin secretagogue according to claim 1 . A food product for promoting the differentiation of preadipocyte cells, comprising the agent for promoting the differentiation of preadipocyte cells according to claim 2 . A feed for promoting adiponectin secretion, comprising the adiponectin secretagogue according to claim 1 . A feed for promoting adipocyte differentiation, comprising the preadipocyte differentiation promoting agent according to claim 2 .

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