JP2012206964A - PPAR-α ACTIVITY REGULATING AGENT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a PPAR-α activity regulating agent which excels in safety and has reduced side effects, preferably, a dual regulating agent for PPAR-α, γ activity which excels in safety and has reduced side effects.SOLUTION: A PPAR-α activity regulating agent which excels in safety and has reduced side effects, preferably, a dual regulating agent for PPAR-α, γ activity which excels in safety, simultaneously activates PPAR-γ activity, and has reduced side effects is obtained by being incorporated with cryptoxanthin included in a mandarin orange or the like. Food and drink, cosmetics and medicines including the regulating agent are provided as well.

Description

  The present invention regulates the activity of PPAR-α, that is, at least one subtype of peroxisome proliferator-responsive receptors (hereinafter abbreviated as PPAR), that is, regulates PPAR-α activity. It relates to the agent.

  PPAR is a nuclear receptor expressed in most vertebrates and controls various biological reactions through expression and control of various genes. PPAR has α, β / δ, and γ subtypes, and the tissues that are expressed and the gene systems that are controlled are different. For example, PPAR-α is highly expressed in muscle and liver and is involved in energy metabolism. In contrast, the expression of PPAR-γ is specific to adipocytes, hardly expressed in other tissues, and is involved in adipocyte differentiation / maturation and production of various cytokines (for example, see Non-Patent Document 1). .)

  The importance of PPAR is that it has been found that metabolic syndrome-related symptoms such as improvement of insulin resistance and serum lipid lowering action are alleviated by the activation of PPAR. For example, thiazolidine derivatives represented by pioglitazone are used as a therapeutic agent for diabetes because they have an action to increase insulin resistance. This action is due to the activation of PPAR-γ, that is, the agonist action of PPAR-γ. (For example, refer nonpatent literature 2). Further, fenofibrate, which is a PPAR-α agonist, has a strong cholesterol and neutral fat lowering action, and thus has been clinically applied as a hyperlipidemic drug (see, for example, Non-Patent Document 3).

  However, administration of a PPAR agonist as described above enhances the expression of PPAR and is not an expression regulation that eliminates abnormal expression in pathological conditions such as obesity and diabetes. For example, in obese model mice, a significant increase in the expression levels of PPAR-α and PPAR-γ in the liver has been reported. However, administration of thiazolidine derivative troglitazone does not reduce or normalize these expression levels. Further enhancement is known (for example, Non-Patent Document 4). That is, since PPAR controls biological functions through the regulation of the expression of various genes as nuclear receptors, further enhancement of abnormal expression increase may have undesirable side effects on the body. .

  For example, the insulin resistance enhancing action of a PPAR-γ agonist is beneficial for diabetics, but the side effect of inducing obesity is also known. Therefore, in order to prevent obesity without impairing the insulin resistance of the PPAR-γ agonist, a method using a PPAR-γ agonist and a PPAR-α agonist in combination has been proposed (see, for example, Patent Document 1). In addition, as a result of searching for substances that control blood glucose levels and serum lipids while preventing obesity with a single agent, a novel carboxylic acid derivative obtained by organic synthesis simultaneously activates PPAR-α and PPAR-γ (2 (Heavier agonist or dual agonist) has been found, and development as a pharmaceutical is underway (see, for example, Patent Document 2).

  While the effects of PPAR agonists have attracted attention, other examples that have been pointed out about the risk include the risk of heart disease of rosigglutazone, which is the thiazolidine derivative, and the risk of developing bladder cancer related to the fenofibrate (for example, non- (See Patent Document 5).

  In response to growing risk concerns due to the excessive effects of PPAR agonists, the US Food and Drug Administration (FDA) has conducted a two-year cancer trial for pharmaceutical companies that are developing PPAR agonists as pharmaceuticals for more than 6 months before starting clinical trials. It is demanded to confirm safety by conducting a primality test (for example, see Non-Patent Document 6).

  As described above, the PPAR agonists known so far, particularly the dual agonists of PPAR-α and γ, have a concern about side effects due to excessive action, so that research and development for practical use is stagnant. However, since the PPAR agonist effect and utility are clear, a safer PPAR activity regulator, particularly a dual regulator, has been desired which has few side effects due to these excessive actions.

Japanese Patent No. 4588448 Japanese Patent No. 4131698

Endocrinology. , 137, 354 (1996) J. et al. Biol. Chem. , 270, p1293-12956 (1995) New Current, 7 (6), 9-19 (1996) Endocrinol, 141 (11), 4021-4031 (2000) N. Engl. J. et al. Med. , 356, 2457-2471 (2007) February 13, 2008 FDA Recommendations Diabetes Melitus: Developing Drugs and Therapeutic Biology for Treatment and Prevention

  An object of the present invention is to provide a PPAR-α activity modulator having excellent safety and less side effects, particularly a dual regulator of PPAR-α and γ activity.

As a result of intensive studies, the present inventors have found that cryptoxanthin contained in mandarin orange and the like regulates PPAR-α activity, and also regulates PPAR-γ activity simultaneously, leading to the present invention.
That is, the gist of the present invention is the following (1) to (6).
(1) A PPAR-α activity regulator comprising cryptoxanthin.
(2) The PPAR-α activity regulating agent according to (1), which has a PPAR-γ activity regulating action.
(3) The PPAR-α activity regulator according to (1) or (2), wherein cryptoxanthin is derived from Unshu oranges.
(4) A food or drink containing the PPAR-α activity regulator according to (1), (2) or (3).
(5) A cosmetic containing the PPAR-α activity regulator according to (1), (2) or (3).
(6) A pharmaceutical comprising the PPAR-α activity regulator according to (1), (2) or (3).

  ADVANTAGE OF THE INVENTION According to this invention, by containing the cryptoxanthin contained in Unshu mikan etc., the PPAR-alpha activity regulator which is excellent in safety and has few side effects, especially the PPAR-alpha activity regulator which regulates PPAR-gamma activity simultaneously. Can be used as foods, drinks, cosmetics and pharmaceuticals by containing the regulator.

It is a graph which shows the body weight fluctuation | variation of the model mouse during the breeding | feeding period which gave various powder feed. It is a graph which shows the amount of various serum lipids of a model mouse after giving various powder feed and raising for 8 weeks. It is a graph which shows the visceral fat mass of the model mouse | mouth after giving various powder feed and raising for 8 weeks. It is a graph which shows the visceral fat area of the model mouse | mouth after giving various powder feed and raising for 8 weeks.

  The present invention will be described in detail below.

  The cryptoxanthin in the present invention is not particularly limited as long as it is cryptoxanthin, and examples thereof include α-cryptoxanthin, β-cryptoxanthin and fatty acid esters thereof. The fatty acid species of the fatty acid ester is not particularly limited, and examples thereof include lauric acid (C12), myristic acid (C14), palmitic acid (C16), and stearic acid (C18).

  Cryptoxanthine, for example, extracted from Wenzhou mandarin oranges, persimmons, papayas, mangoes, etc. is preferably used, and among them, it can be eaten safely with traditional Japanese fruits, has a high production amount, and contains cryptoxanthin content In particular, those made from Wenzhou mandarin orange are preferred.

  In Satsuma mandarin, not only raw fruits but also processed Satsuma mandarin and its intermediates can be used as raw materials. Examples of processed products and intermediates thereof include Unshu mandarin orange juice, residue after squeezing juice from Unshu mandarin orange, dried residue, enzyme-treated residue, solvent extract from Unshu mandarin orange, and the like. Among them, the enzyme-treated product of Wenzhou mandarin orange residue in which an enzyme such as cellulase was allowed to act on the residue reduced the residue by solubilizing and removing the dietary fiber contained in a large amount in the residue, and the relative concentration of cryptoxanthin Is suitable as a raw material. In addition, the dried product of the enzyme-treated product of Unshu mandarin orange juice residue is more suitable because the concentration of cryptoxanthin increases.

  The squeezed residue of Unshu mandarin orange and the enzyme-treated product of the residue may be used as they are as the PPAR-α activator of the present invention, or those that have undergone some processing may be used. Specifically, a residue obtained by solid-liquid separation of these, a product obtained by drying a residue obtained by solid-liquid separation, a product obtained by drying the reactant as it is without solid-liquid separation, or the like may be used as the PPAR-α activator. In addition, the water washing method of adding and stirring water to the residue of squeezed mandarin orange juice, the enzyme-treated product of the residue itself, or the residue after solid-liquid separation, followed by solid-liquid separation again It is preferable because water-soluble impurities such as acid and sugar present in the residue and the enzyme-treated product can be easily removed.

  The solvent extract of Unshu mandarin orange is obtained by extracting a component containing cryptoxanthin from Unshu mandarin orange or its enzyme-treated product using a solvent, supercritical carbon dioxide or the like. As the solvent used for extraction, any solvent can be used as long as β-cryptoxanthin, which is a functional component, can be extracted from Unshu orange, which is a raw material, or a processed product thereof. One kind of solvent may be used alone, or a plurality of solvents may be mixed and used. Examples of those that satisfy these conditions include alcohols such as methanol, ethanol, propanol and butanol, polyhydric alcohols such as ethylene glycol, propylene glycol, butylene glycol and glycerin, ketones such as acetone and methyl ethyl ketone, methyl acetate and acetic acid. Esters such as ethyl, ethers such as tetrahydrofuran and diethyl ether, halogenated hydrocarbons such as dichloromethane, dichloroethane and chloroform, aliphatic hydrocarbons such as hexane and pentane, aromatic hydrocarbons such as toluene, polyethylene glycol Polyethers such as pyridine, pyridines and the like can be used. Of these, ethanol is preferable because of its high extraction efficiency of cryptoxanthin. Moreover, when extracting with these organic solvents, in order to raise extraction efficiency, additives, such as water and surfactant, can be added in the range which does not impair the effect of this invention.

  Since the purity of cryptoxanthin can be increased following the extraction, concentration, desalting, partition purification, column chromatography, and the like can be used. A process for changing the shape of the extract, such as powdering or emulsification, can also be used. Furthermore, not only these methods are performed alone, but a plurality of methods may be combined.

  In the present invention, modulation of PPAR-α activity refers to activating PPAR-α activity (ie, exhibiting PPAR-α agonist activity) or suppressing PPAR-α activity (ie, PPAR-α antagonist activity). Simultaneously regulating means that both PPAR-α activity and PPAR-γ activity are activated simultaneously in the same living tissue (dual agonist effect), or that both are inhibited simultaneously (dual antagonist) Action).

  The amount of cryptoxanthin in the PPAR-α activity regulator in the present invention is not particularly limited. For example, it is preferably ingested so that the daily intake of cryptoxanthin is 0.01 to 5000 μg. It is more preferable to ingest so that it may become -2000 micrograms.

  The food / beverage products of this invention say what is ingested orally, such as a foodstuff, a drink, a favorite product, and a supplement which mix | blended the PPAR- (alpha) activity regulator of this invention. Forms such as food products and beverages are not particularly limited, and can be main dishes such as breads and noodles, cheese, ham, wiener, processed food products such as seafood, fruit juice drinks, carbonated drinks, It can be a beverage such as a milk beverage, or a favorite product such as a cookie, cake, jelly, pudding, candy, or yogurt. The form when used as a supplement or the like is not particularly limited, and examples thereof include tableted products (tablets), hard capsules, soft capsules, fine powders, granules, and the like, as well as troches, chewable tablets, and sheet agents. In addition to solid forms, liquid drinks and semi-solid soft jelly forms are also included.

  The form of the cosmetic product of the present invention is not particularly limited, and in view of the nature of the present invention, the use as an inner cosmetic product that is taken orally rather than externally is more preferable. Examples of the form of the inner beauty product include tableted products (tablets), hard capsules, soft capsules, fine powders, granules, and the like, as well as troches, chewable tablets, and sheet agents. In addition to solid forms, liquid drinks and semi-solid soft jelly forms are also included.

  The form of the pharmaceutical product of the present invention is not particularly limited, and examples thereof include tableted products (tablets), hard capsules, soft capsules, fine powders, granules, and the like, as well as troches, chewable tablets, and sheet agents. In addition to solid forms, liquid drinks and semi-solid soft jelly forms are also included.

  You may make the food-drinks, cosmetics, and pharmaceutical of this invention contain another functional component in the range which does not impair functionality. For example, vitamins such as vitamins A, B, C, and E, and carotenoids such as β-carotene, lycopene, and astaxanthin, anthocyanins, catechins, and isoflavones can be included.

  The amount of cryptoxanthin contained in the food / beverage products, cosmetics and pharmaceuticals of the present invention varies depending on the form of the composition. For example, 10 g / g to 10 g of cryptoxanthin per 100 g of food / beverage products, cosmetics and pharmaceuticals. It is preferable. Among these, from the viewpoint of economy and effects, 100 pg to 1 g is more preferable, and 1 ng to 500 mg is more preferable.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

  Cryptoxanthine content in various samples was connected to a Waters Resolve C18 (φ3.9 × 150 mm) column using Shimadzu High Performance Liquid Chromatography (HPLC) LC-10A, and water: methanol (volume ratio) = A sample dissolved 50/50 was injected. As a mobile phase, methanol: ethyl acetate (volume ratio) = 7: 3, a column temperature of 30 ° C., a flow rate of 1.0 ml / min, and a detection wavelength of 450 nm were measured. The cryptoxanthin concentration was calculated from the absorbance of an unknown sample by preparing a calibration curve using the EXTRASYNTHESE product as a standard sample.

Example 1
Sumiteam PX (manufactured by Shin Nippon Chemical Industry Co., Ltd., pectinase 5,000 units / g, arabanase 90 units / kg) on 1 kg of Wenzhou mandarin orange juice residue (mandarin orange juice, moisture content of about 90% by mass) g) 1 g and 1 g of cellulase Y-NC (manufactured by Yakult Pharmaceutical Co., Ltd., cellulase 30,000 units / g) which is an enzyme agent of cellulase / hemicellulase were added, stirred well, and allowed to stand at room temperature for 8 hours. . After centrifuging the reaction solution to remove the supernatant, water was added and stirred, and the supernatant was again removed by centrifugation. This precipitate was dried with a freeze dryer, and after pulverizing for 5 minutes with a knife type pulverizer (Retsch, GM200), 50 g of a pulverized product (composition 1) passing through a 300-mesh sieve was obtained. The β-cryptoxanthin concentration in Composition 1 was 1 mg / g in terms of free form.

Example 2
500 ml of ethanol was added to 50 g of the composition 1, and the filtrate obtained by filtering and removing the solid content after stirring for 2 hours at room temperature was concentrated to obtain 20 g of a concentrate (composition 2). The β-cryptoxanthin concentration in Composition 2 was 2.5 mg / g in terms of free form.

Example 3
1 g of composition 2 was mixed and dispersed with 1 L of milk (Meiji Dairies, Meiji Tasty Milk) together with 5 mg of an emulsifier (manufactured by Mitsubishi Chemical Co., Ltd., polyglyceric acid ester) to produce a cryptoxanthin-containing milk beverage (milk) (Contains 0.25 mg of β-cryptoxanthin in 100 g of beverage).

Example 4
2.5 kg of composition 1 was mixed with 1 kg of yogurt (Meiji dairy industry, Meiji Bulgaria yogurt) to produce a cryptoxanthin-containing yogurt (containing 0.25 mg of β-cryptoxanthin in 100 g of yogurt).

Example 5
Soft capsules were produced by the following production method.
Ingredient Compounding amount (in 100g)
1) Composition 1 50 g
2) Vitamin E 5g
3) Vitamin C derivative 5g
4) Olive oil 40g
Production methods: 1) to 4) were uniformly mixed, and then filled into a soft capsule packaging material made of gelatin, beeswax or the like at 200 mg / tablet.

Example 6
A tablet was produced by the following production method.
Ingredient Compounding amount (in 100g)
1) Composition 2 5000 mg
2) Maltitol 50.0g
3) Crystalline cellulose 30.0g
4) Sucrose fatty acid ester 5.0 g
5) Vitamin C 10.0g
6) Sweeteners Appropriate amount 7) Acidulant Appropriate amount 8) Fragrance Appropriate amount Manufacturing method: 1) to 8) were uniformly mixed and granulated by a conventional method, and then tableted at 0.5 g / tablet.

Example 7
A drink was produced by the following production method.
Ingredient Compounding amount (in 100 mL)
1) Composition 2 200 mg
2) Honey 320mg
3) 600 mg of cyclic oligosaccharide
4) Sweetener appropriate amount 5) Acidulant appropriate amount 6) Preservative appropriate amount 7) Perfume appropriate amount 8) Water 1) to 7) were sequentially added to the remaining manufacturing method: 8) to make 100 mL.

<Test Example 1>
TSOD (Tsumura Suzuki Obese, Diabetes), which is an obese model mouse, is fed with a diet obtained by adding composition 1 to a powdered feed (trade name: CRF-1, manufactured by Charles River Japan) so that the composition ratio is 10% by mass. Serum, visceral fat, liver, and thigh muscle were collected after 8 weeks of breeding while monitoring body weight. The collected serum was measured for cholesterol, neutral fat, and free fatty acid. Visceral fat was weighed, formalin-fixed and HE-stained to prepare a specimen, and the adipocyte area was compared. In addition, RNA was extracted from visceral fat, liver and thigh muscles, and gene expression was compared by DNA microarray method.

<Comparative Example 1>
TSOD, which is an obese model mouse, is fed with a diet obtained by adding 10% by mass of carboxymethylcellulose to a powdered feed (trade name: CRF-1, manufactured by Charles River, Japan), and raised for 8 weeks while monitoring the body weight, then serum, visceral fat The liver and thigh muscles were collected. Samples such as collected serum were analyzed in the same manner as in Test Example 1 and compared with Test Example 1.

<Comparative example 2>
A diet obtained by adding 10% by mass of carboxymethylcellulose to powdered feed (trade name: CRF-1, manufactured by Charles River, Japan) to TSNO (Tsumura Suzuki Non-Obese, Diabetes), which is a genetically-controllable normal mouse of an obese model mouse Serum, visceral fat, liver, and thigh muscle were collected after 8 weeks while monitoring the body weight. Samples such as collected serum were analyzed in the same manner as in Test Example 1.

<Result 1. Weight>
In Comparative Example 1 (TSOD mouse / normal feed), the body weight significantly increased compared to Comparative Example 2 (TSNO mouse / normal feed). On the other hand, in Test Example 1 (TSOD mouse / Composition 1 supplemented feed), the body weight at the initial stage of the breeding was equivalent to that of Comparative Example 1, but the weight gain was suppressed after the second week of breeding. The value was significantly lower than that (FIG. 1). In addition, there was no difference in the food intake of Test Example 1 and Comparative Example 1.

<Result 2. Serum lipid>
Serum lipids (cholesterol, neutral fat, advantageous fatty acid) in Comparative Example 1 were significantly increased as compared with Comparative Example 2, but serum lipids in Test Example 1 were significantly decreased as compared with Comparative Example 1. (FIG. 2).

<Result 3. Visceral fat>
The visceral fat weight (FIG. 3) and area (FIG. 4) of Comparative Example 1 were significantly increased as compared to Comparative Example 2, but were significantly decreased in Test Example 1 as compared to Comparative Example 1.

<Result 4. DNA Microarray>
The expression levels of PPAR-α and PPAR-γ in Test Example 1, Comparative Example 1, and Comparative Example 2 were measured by the DNA microarray method, and the expression level of Comparative Example 2 (feeding β-cryptoxanthin-free feed to TSNO mice) Table 1 shows relative amounts of As a result, the expression levels of PPAR-α and PPAR-γ in Comparative Example 1 (providing TSOD mice with β-cryptoxanthin-free feed) increased in the liver and decreased in visceral fat and muscle. However, in Test Example 1 (feeding β-cryptoxanthin-containing feed to TSOD mice), PPAR-α and PPAR-γ in the liver decreased and PPAR-α and PPAR-γ in visceral fat and muscle increased compared to Comparative Example 2. Was. That is, β-cryptoxanthin acted as a dual antagonist in the liver of TSOD mice and as a dual agonist in visceral fat and muscle.
These changes indicate that β-cryptoxanthin modulates abnormal PPAR expression in obese mice in a direction that normalizes rather than potentiates it, and this regulatory function is related to the obesity of β-cryptoxanthin. It is thought to be a cause of the inhibitory effect.

From the above results, various obesity parameters such as body weight, visceral fat reduction, serum lipid reduction, visceral fat cell hypertrophy suppression, etc. showed a tendency to normalize by ingestion of composition 1, ie, β-cryptoxanthin . On the other hand, since both PPAR-α expression and PPAR-γ expression were changed to suppress obesity, β-cryptoxanthin acts as a dual regulator of PPAR-α and PPAR-γ, and TSOD mice It is thought that obesity was suppressed.

Claims (6)

A PPAR-α activity regulator comprising cryptoxanthin. 2. The PPAR-α activity regulator according to claim 1, which has a PPAR-γ activity regulating action. The PPAR-α activity regulator according to claim 1 or 2, wherein cryptoxanthin is derived from Unshu mandarin oranges. Food-drinks containing the PPAR- (alpha) activity regulator of Claim 1, 2, or 3. Cosmetics containing the PPAR-α activity regulator according to claim 1, 2 or 3. The pharmaceutical containing the PPAR- (alpha) activity regulator of Claim 1, 2, or 3.