JP2010189291A - Adiponectin increasing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adiponectin increasing agent that exhibits an excellent expression-enhancing action for adiponectin. <P>SOLUTION: The adiponectin increasing agent includes paprika coloring matter as an effective ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel adiponectin increasing agent.

  Adiponectin is a factor (adipokine) secreted mainly from adipocytes, but has recently been reported to have an insulin resistance improving action and an anti-arteriosclerotic action (Non-patent Document 1). As adipokines other than adiponectin, TNF-α (tumor necrosis factor α), resistin, free fatty acid, PAI-1 (plasminogen activator inhibitor 1), etc. are known, but these all cause insulin resistance. It has become clear. Adiponectin, which is a good adipokine that improves insulin resistance, unlike many adipokines, has recently received much attention.

  It has been shown as clinical data that not only the action of adiponectin but also a decrease in blood adiponectin level is a predictive marker for the onset of diabetes (Non-patent Document 2). In addition, it has been reported that the amount of adiponectin in the blood shows an inverse correlation with the degree of obesity (Non-patent Document 3). Is presumed to play an important role. Indeed, it has been shown that the lower the blood adiponectin, the higher the risk of developing myocardial infarction. More recently, it has been reported that the amount of adiponectin in blood has a positive correlation with bone density (Non-patent Document 4), and it has been suggested that an increase in adiponectin can prevent and improve a decrease in bone density.

  Several in vitro test systems for evaluating the expression level of adiponectin have been established. For example, a system in which 3T3-L1 cells, which are adipose precursor cell lines, are differentiated is known. In this system, it has been reported that the expression level of adiponectin decreases by adding TNF-α, insulin, dexamethasone which is a glucocorticoid receptor agonist, etc. (Non-patent Document 5). In addition, it has been reported that the addition of glucocorticoid also reduces the expression level of adiponectin, while increasing the secretion of PAI-1, which is a bad adipokine (Non-patent Document 6). Furthermore, it has been reported that blood adiponectin is decreased by administration of glucocorticoid in human clinical trials (Non-patent Document 7). From this, it is considered that glucocorticoid itself causes an adipokine abnormality and affects the expression and secretion of adiponectin.

  Glucocorticoids have also been shown to be involved in adipocyte dysfunction in obesity. HSD1 (11β-hydroxysteroid dehydrogenase 1) is an enzyme that converts inactive glucocorticoid to cortisol, but there is a positive correlation between obesity and HSD1 expression in adipose tissue. It has become clear (Non-Patent Document 8). Furthermore, it is known that knockout mice of HSD1 are unlikely to develop metabolic syndrome (Non-patent Document 9), suggesting that glucocorticoids overproduced by adipocytes are one of the causes of metabolic syndrome. Yes.

  Based on the above, in in vitro test systems using adipocytes, substances that increase the expression level of adiponectin even when dexamethasone or glucocorticoid is added are useful as substances that increase adiponectin expression. It is considered useful for the prevention and improvement of various diseases such as hypertension, metabolic syndrome located upstream of various lifestyle-related diseases, and reduction of bone density.

  On the other hand, paprika pigment is an oil-soluble pigment composed mainly of capsanthin obtained from pepper fruit, and is widely used as a natural pigment in foods and the like. Capsanthin is a component belonging to carotenoids, and carotenoids have been reported to have a preventive / therapeutic effect on diseases caused by a low adiponectin state (Patent Document 1), but carotenoids have an action to increase adiponectin expression. There is no data to show that. Patent Document 1 does not exemplify capsanthin, and so far there is no known effect of paprika pigment on adiponectin.

JP 2005-232059 A

Kadowaki, T. et al .: J. Clin. Invest., 116: 1784.1792 (2006). Spranger, J. et al .: Lancet., 361: 226-228 (2003). Arita, Y. et al .: Biochem. Biophys. Res. Commun., 257: 79-83 (1999). Richards, JB. Et al .: J. Clin Endocrinol Metab., 92: 1517-23 (2007) Fasshauer. Et al .: Biochem. Biophys. Res. Commun. 290: 1084-1089 (2002). Ayachi S EI., Et al .: J Thromb Haemost., 4: 621-627 (2006) Fallo F., et al .: Eur J Endocrinol., 150: 339-344 (2004) Masuzaki H., et al .: J Jpn Coll Angiol, 46: 345-351 (2006) Kotelevtsev Y, et al .: Proc Natl Acad Sci USA, 94: 14924-14929 (1997)

  The present invention relates to providing an adiponectin increasing agent having an excellent adiponectin expression increasing action.

As a result of studying substances having an adiponectin expression increasing action, the present inventors have found that paprika pigment can effectively increase adiponectin expression in blood and is useful as an adiponectin increasing agent.
As described above, capsanthin, which is a main component of paprika pigment, is a component belonging to carotenoids, but carotenoids exemplified in Patent Document 1, such as β-carotene, did not have an action to increase adiponectin expression. . Similarly, the actinone yellow pigment mainly composed of crocetin belonging to the same carotenoids as capsanthin and β-carotene did not show an adiponectin expression increasing action. In addition, α-tocopherol, which has higher antioxidant power than capsanthin, was also not found to increase the adiponectin expression. Therefore, it is completely unexpected that the above effect is achieved by the paprika pigment.

  That is, the present invention provides an adiponectin increasing agent containing a paprika pigment as an active ingredient.

  By using the adiponectin increasing agent of the present invention, it is possible to increase the expression level of adiponectin in the blood, and various diseases that are considered to be useful in increasing the expression thereof, such as hypoadiponectinemia, impaired glucose tolerance, Diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia Prevention, improvement such as symptom, hypercholesterolemia, obesity, metabolic syndrome, decrease in bone density.

It is a figure which shows the adiponectin expression increase effect of the paprika pigment | dye in an adipocyte. It is a figure which shows the plasma adiponectin expression increase effect of the paprika pigment | dye in a REM sleep inhibition system.

  The paprika pigment used in the present invention is a pigment obtained from the fruit of Capsicum annuum Linne, and has capsanthin as a main component. Capsicum is also called alias paprika and sweet pepper, and paprika pigment is also called capsicum pigment, capsicum pigment, Paprika oleoresin, carotenoid, carotenoid pigment, carotenoid, carotenoid pigment, and is marketed under those names. Paprika pigments are widely used in foods as food additives and as coloring agents.

The paprika pigment is an extract obtained by extracting a paprika fruit as it is or after being dried and then cut into a suitable size or pulverized, its diluted solution, its concentrated solution, its dried powder or Paste forms and the like are included. The extraction method may be any of immersion, decoction, leaching, reflux extraction, supercritical extraction, ultrasonic extraction, microwave extraction, and the like.
As the extraction solvent, either a polar solvent or a nonpolar solvent can be used, and these can also be mixed and used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran Linear and cyclic ethers such as diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; benzene and toluene Aromatic hydrocarbons such as; pyridines; supercritical carbon dioxide; oils, waxes, oils such as rapeseed oil, olive oil, soybean oil, etc., and these can be used alone or in combination of two or more. It is also possible to carry out repeatedly changed. Of these, ethanol, hexane, supercritical carbon dioxide, fats and oils, and the like are preferably used.
For extraction, for example, 1 to 50 parts by mass of a solvent is used per 1 part by mass of the pepper, and it is preferably immersed or heated to reflux at 0 to 100 ° C. for several hours to several weeks.

  The paprika pigment used in the present invention may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention. Further, the obtained crude product is publicly known. These separation and purification methods may be combined as appropriate to increase their purity. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph and the like.

The adiponectin increasing agent of the present invention showed an excellent adiponectin expression increasing action in an in vitro test system using lipocytes and a REM sleep inhibition system, as shown in Examples below.
The effect of increasing adiponectin expression is an indicator of whether the expression level of adiponectin increases in the in vitro test system using adipocytes even under the influence of glucocorticoid, which is thought to suppress the expression and secretion of adiponectin, as described above. Can be evaluated as In addition, in the REM sleep inhibition system, the improvement of blood adiponectin that has been reduced by the inhibition of REM sleep in rats can be evaluated as an index. Therefore, the adiponectin-increasing agent of the present invention is useful for various diseases considered to be useful for increasing adiponectin secretion, such as hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications. , Hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia, obesity, metabolic syndrome, bone density It can be used as a preventive or ameliorating agent (hereinafter referred to as “adiponectin increasing agent, etc.”), and can be used to produce these agents.

  Such adiponectin increasing agents include, for example, hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular disease For humans or animals that exhibit various effects such as prevention or improvement of cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia, obesity, metabolic syndrome, bone density reduction It can be used as pharmaceuticals, quasi drugs, foods, and functional foods. The adiponectin increasing agent includes, for example, hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular The concept is to prevent or improve disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia, obesity, metabolic syndrome, and decrease in bone density. It can be applied to the displayed food, functional food, food for the sick, and food for specified health use.

  When using the adiponectin increasing agent of this invention as a pharmaceutical, it can be administered with arbitrary dosage forms. Examples of the dosage form include oral, enteral, transmucosal, injection and the like. Examples of the dosage form of the preparation for oral administration include tablets, capsules, granules, powders, and syrups. Examples of parenteral administration include intravenous injection, intramuscular injection, suppository, inhalation agent, transdermal absorption agent, eye drop, nasal drop and the like.

  In such a preparation, the paprika pigment of the present invention may be used in combination with a pharmaceutically acceptable carrier. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet rays. Absorber, moisturizer, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, corrigent, flavoring agent, extender, surfactant, dispersant, buffer, preservative, fragrance, coating agent Etc.

Of these dosage forms, oral administration is preferred, and when used as a preparation for oral administration, the content of the paprika pigment of the present invention in the preparation is usually 0.005 to 5% by mass of the total mass of the formulation, It is preferable that it is 0.01-1 mass%.
Note that the content of the paprika pigment is calculated as the weight of the extract (after extraction, after removal of the solvent; the same applies hereinafter).

  The dosage of the above formulation may vary according to the patient's condition, body weight, sex, age or other factors, but the daily dosage per adult for oral administration is usually paprika dye (extract weight 1-2000 mg, 10-1000 mg, and 50-500 mg are more preferable. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times a day.

  When the adiponectin increasing agent of the present invention is used as a food, the form thereof may be solid, semi-solid or liquid. Examples of food include breads, noodles, confectionery, jelly, dairy products, frozen foods, instant foods, processed starch products, processed meat products, other processed foods, beverages, soups, seasonings, nutritional supplements, etc. , And their raw materials. Further, like the above-mentioned oral administration preparation, it may be in tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form and the like.

  To prepare various forms of food, paprika pigments can be used alone or with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorbers, oxidation An inhibitor, a humectant, a thickener and the like can be used in appropriate combination.

  Moreover, although the content of the paprika pigment | dye (extract weight conversion) in a foodstuff changes with the usage forms, it is 0.001-2 mass% normally in the form of a drink, and is 0.002-1 mass% normally. Is preferable, and 0.01-0.5 mass% is more preferable. Moreover, in solid food forms, such as a tablet and processed food, it is 0.01-20 mass% normally, 0.02-10 mass% is preferable, and 0.1-5 mass% is more preferable.

Example 1
[Test method]
This was performed in a system in which 3T3-L1 cells (ATCC), which is a mouse fat precursor cell line, were differentiated into adipocytes. The basic medium was DMEM (high glucose, Invitrogen 11965-092). The 3T3-L1 cells were seeded at 7.5 x 10 ³ cell / well in 24-well plates were cultured in 4-day regular medium (10% fetal calf serum / DMEM), differentiation medium 1 (1 [mu] M insulin, 0.5 mM isobutyl 3 days in methylxanthine, 0.1 μM dexamethasone, 10% fetal bovine serum / DMEM), 3 days in differentiation medium 2 (1 μM insulin, 10% fetal bovine serum / DMEM), and 3-6 in normal medium Cultured for days.
After exchanging with DMEM medium excluding fetal bovine serum and culturing for 6 hours, samples (Paprika Dye; “Paprika Base 150” Saneigen FFI (contains 20% as Paprika Dye)), Carrot Carotene; Sankyo Lifetech, Gardenia S. elegans; San-Eigen FFI, α-tocopherol; Thereafter, RNA was extracted from the collected cells using an RNA extraction kit (RNeasy mini kit [Qiagen]). The extracted total RNA was subjected to reverse transcription reaction (37 ° C., 1 hour) with oligo dT primer (Invitrogen) and MMLV RT (Invitrogen) to prepare cDNA. The adiponectin mRNA expression level of the prepared cDNA was measured by Real-Time PCR method using TaqMan (registered trademark) probe. The expression level was corrected with the GAPDH gene level. TaqMan (registered trademark) probes and primers used were TaqMan (registered trademark) Gene Expression Assays (ABI, AssayID; Mm00456425_m1) for adiponectin and 4352339E (ABI) for GAPDH. The results are shown as relative values when the control group is 100%. The values in the graph are shown as mean ± standard error.

[result]
In the control group to which dexamethasone, which is a glucocorticoid receptor agonist, was added, the adiponectin mRNA expression level was lower than that in the group to which dexamethasone was not added.
In contrast, in the group to which paprika dye (6 μg / ml) was added, the adiponectin mRNA expression level was significantly increased compared to the control group. However, in the group added with carrot carotene, which is mainly composed of β-carotene, which belongs to the same carotenoids as capsanthin, which is the main component of paprika pigment, and in the group added with gardenia yellow, which is mainly composed of crocetin, 100 μg / ml Even when added at a dose, the adiponectin mRNA expression level did not increase at all, and even when α-tocopherol (20 μM), a vitamin having an antioxidant action, was added, the adiponectin mRNA expression level did not increase at all (FIG. 1).

Example 2
[Test method]
The increase effect of blood adiponectin in animals with paprika pigment was evaluated by REM sleep inhibition system using rats. In this evaluation system, water is placed in the cage and a small cylindrical platform (platform) on which rats can ride is installed. Rats can rest on the platform, but when they go to REM sleep, their muscles relax and their posture collapses and touches the water surface. Therefore, although the rat can take non-REM sleep, it is in a state where it cannot take REM sleep. In this REM sleep inhibition system, blood adiponectin decreases in a short period of time, and the decrease is suppressed if the substance has an adiponectin expression increasing action. It has been confirmed that this system can evaluate an adiponectin increasing agent (Japanese Patent Application No. 2008-107047).
SD rats (♂, 10-11 weeks old) were grouped to equal body weight. A cylindrical platform with a diameter of 6.0 cm and a height of 2.5 cm, in which the rat can rest, was placed in the cage, and water was added up to 1 cm below the platform. This cage was raised for 2 days (1 animal / 1 cage). The resting group was reared in a cage with a normal flooring. The paprika pigment ("Paprika Base 150" Saneigen FFI) administration group was orally administered with a sonde twice a day at a dose of 60 mg / kg (120 mg / kg / day) as a paprika pigment. The paprika pigment was administered for a total of 3 days from 1 day before the start of REM sleep inhibition. Distilled water was administered to the control group, and the resting group was raised in cages with ordinary bedding, and distilled water was administered (each group N = 6). Immediately after the end of REM sleep inhibition, blood was collected from the abdominal aorta into a Venoject vacuum blood collection tube under anesthesia with Foren (Dainippon Pharmaceutical Co., Ltd.), and the resulting heparin plasma was stored at −80 ° C. until use. Plasma adiponectin was measured with a mouse / rat adiponectin ELISA kit (Otsuka Pharmaceutical). After the plasma was diluted 1111 times with the dilution buffer in the kit, the adiponectin concentration was measured according to the kit protocol. The values in the graph are shown as mean ± standard error.

[result]
Compared to the resting group, plasma adiponectin decreased significantly in the control group loaded with REM sleep inhibition. On the other hand, plasma adiponectin increased significantly in the paprika dye administration group compared to the control group (FIG. 2).
From this result, it was confirmed that the adiponectin increasing agent of the present invention can significantly increase the expression of adiponectin in blood.

Claims (1)

  Adiponectin increasing agent containing paprika pigment as an active ingredient.