JP2008266311A - Adiponectin increasing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new adiponectin increasing agent and a preventive or improving agent of obesity or diseases caused by obesity. <P>SOLUTION: The adiponectin increasing agent including effective components of a phospholipid composition containing 50 wt.% or more of phosphatidylserine, phosphatidylinositol and phosphatidic acid in total, and 40 wt.% or more of phosphatidylserine and phosphatidylinositol in total. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an agent for increasing blood adiponectin, and an agent for preventing or improving obesity or diseases caused by obesity.

  Adiponectin is a hormone factor produced specifically in adipose tissue, and mainly acts on skeletal muscle, liver, and arterial wall cells to promote fat metabolic degradation and sugar uptake. Due to this action, it is known that adiponectin exhibits an antidiabetic action, an antiarteriosclerotic action, an antihypertensive action and the like.

  As visceral fat increases, the amount of adiponectin produced from adipose tissue decreases, and this hypoadiponectinemia promotes obesity. Hypoadiponectinemia is considered to cause various diseases caused by obesity such as diabetes, hypertension, hyperlipidemia, and arteriosclerosis. Therefore, increased production of adiponectin has attracted attention in recent years as it leads to the prevention or treatment of obesity, various diseases caused by obesity, and lifestyle-related diseases.

  Patent Document 1 discloses a liver function improving agent containing phosphatidylserine as an active ingredient, which is effective for prevention, improvement and treatment of various viral, alcoholic, and drug-related liver dysfunctions.

In Patent Document 2, since blood cholesterol is reduced by administering a phospholipid composition containing phosphatidylinositol at a high concentration to rats, this mixture is used as a blood neutral fat and blood cholesterol metabolism-improving agent. It is described that it is suitable.
Japanese Patent Laying-Open No. 2005-112731 JP 2000-300196 A

  An object of the present invention is to provide a novel adiponectin elevating agent and an agent for preventing or improving obesity or diseases caused by obesity.

  As a result of intensive studies to solve the above problems, the present inventors have found that the total ratio of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 50% by weight or more, and the total ratio of phosphatidylserine and phosphatidylinositol is 40%. Discovered the effect that a phospholipid composition with a weight percent or more increases the concentration of adiponectin in the blood, thereby making this phospholipid composition suitable as an active ingredient for prevention or amelioration of obesity and various diseases caused by obesity Found that can be used.

The present invention has been completed based on the above findings, and provides novel adiponectin elevating agents and the like of the following items.
Item 1. An adiponectin elevating agent comprising as an active ingredient a phospholipid composition in which the total ratio of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 50% by weight or more, and the total ratio of phosphatidylserine and phosphatidylinositol is 40% by weight or more.
Item 2. Item 2. The adiponectin elevating agent according to Item 1, wherein the ratio of phosphatidylserine to the total of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 30% by weight or more.
Item 3. Item 3. The adiponectin increasing agent according to Item 1 or 2, wherein a ratio of phosphatidylinositol to phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 40% by weight or less.
Item 4. Item 4. The adiponectin elevating agent according to any one of Items 1 to 3, wherein the phospholipid composition contains 20 to 50% by weight of phosphatidylserine and 10 to 25% by weight of phosphatidylinositol relative to the whole.
Item 5. Caused by obesity or obesity, comprising as an active ingredient a phospholipid composition in which the total ratio of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 50% by weight or more, and the total ratio of phosphatidylserine and phosphatidylinositol is 40% by weight or more Preventive or ameliorating disease.
Item 6. Item 6. The agent for preventing or improving obesity or a disease caused by obesity according to Item 5, wherein the ratio of phosphatidylserine to the total of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 30% by weight or more.
Item 7. Item 7. The agent for preventing or improving obesity or a disease caused by obesity according to Item 5 or 6, wherein the ratio of phosphatidylinositol to the total of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 40% by weight or less.
Item 8. Item 5. The obesity according to any one of Items 5 to 7, wherein the phospholipid composition contains 20 to 50% by weight of phosphatidylserine and 10 to 25% by weight of phosphatidylinositol based on the whole of the phospholipid composition. Preventive or ameliorating agent.
Item 9. The prevention of the disease caused by obesity or obesity according to any one of Items 5 to 8, wherein the disease caused by obesity is diabetes, hyperlipidemia, hypertension, arteriosclerosis, liver dysfunction, or metabolic syndrome Or an improving agent.

  The adiponectin elevating agent of the present invention and the preventive or ameliorating agent for obesity or diseases caused by obesity are a total ratio of phosphatidylserine, phosphatidylinositol, and phosphatidic acid, and a total ratio of phosphatidylserine and phosphatidylinositol. Is characterized in that it contains a phospholipid composition having an amount of 40% by weight or more as an active ingredient. Adiponectin is a kind of hormone secreted from adipose tissue, and the phospholipid composition of the present invention has an effect of increasing the blood concentration of this adiponectin.

When the adiponectin concentration in the blood increases, fat metabolism and sugar uptake in the liver and skeletal muscle are promoted, and as a result, obesity and diseases caused by obesity are improved.
Specifically, adiponectin acts on the liver and skeletal muscle to activate AMP kinase that promotes sugar uptake and fatty acid combustion. Activated AMP kinase suppresses gluconeogenesis in the liver and burns fat, and skeletal muscle takes in sugar by burning sugar. As a result, obesity is improved. In addition, in the liver, neutral fat and cholesterol are decreased to reduce fat, and obesity to liver function and fatty liver are improved. Moreover, when the concentration of adiponectin in the blood increases, the concentration of neutral fat and cholesterol in the blood decreases, and hyperlipidemia, hypertension, and arteriosclerosis are improved. In addition, gluconeogenesis in the liver is suppressed, and sugar in the blood is taken into skeletal muscle, thereby improving diabetes.
Moreover, since it becomes difficult to become obese when the blood adiponectin concentration increases, obesity and diseases caused by obesity are prevented.

  Adiponectin is secreted in an appropriate amount from normal adipose tissue, but it tends to decrease in obese adipose tissue. Therefore, the agent of the present invention can be suitably used for humans whose secretion amount of adiponectin is reduced due to obesity. Genetically, males have a lower secretion amount of adiponectin than females, so males are particularly suitable.

Hereinafter, the present invention will be described in detail.
Phospholipid composition The adiponectin elevating agent of the present invention and the preventive or ameliorating agent for obesity or diseases caused by obesity are phosphatidylserine (hereinafter sometimes referred to as “PS”), phosphatidylinositol (hereinafter referred to as “PI”). A phospholipid composition in which the total weight ratio of phosphatidic acid (hereinafter sometimes referred to as “PA”) is 50% or more and the total weight ratio of phosphatidylserine and phosphatidylinositol is 40% or more. It is characterized by including as an ingredient.

In the adiponectin elevating agent and the agent for preventing or ameliorating a disease caused by obesity according to the present invention, the total content of PS, PI and PA is preferably 55% by weight or more based on the entire phospholipid composition, 70% by weight or more is more preferable. If it is the said range, the adiponectin raise effect will fully be acquired. The upper limit of the total content of PS, PI and PA is usually about 80% by weight from the viewpoint of the raw material composition.
Moreover, 45 weight% or more is preferable with respect to the whole phospholipid composition, and, as for the total content of PS and PI, 60 weight% or more is more preferable. If it is the said range, the adiponectin raise effect will fully be acquired. The upper limit of the total content of PS and PI is usually about 70% by weight from the viewpoint of the raw material composition.
The ratio of PS to the total of PS, PI, and PA is preferably 30% by weight or more, and more preferably 45% by weight or more. The upper limit of the ratio of PS is usually about 70% by weight.
Further, the ratio of PI to the total of PS, PI, and PA is preferably 40% by weight or less, and more preferably 30% by weight or less. The lower limit of the ratio of PI to the total of PS, PI and PA is usually about 20% by weight.
The phospholipid composition preferably contains about 20 to 50% by weight of PS and about 10 to 25% by weight of PI, about 25 to 45% by weight of PS, and 15 to 20% by weight of PI. It is more preferable to include about%.

  In addition to the acidic phospholipids of PS, PI, and PA, the phospholipid composition includes phosphatidylcholine (hereinafter also referred to as “PC”), phosphatidylethanolamine (hereinafter also referred to as “PE”). Or a neutral phospholipid.

Formulation

  The adiponectin elevating agent and the agent for preventing or ameliorating a disease caused by obesity according to the present invention can be administered orally or parenterally. Examples of orally administered agents include solid preparations such as powders, granules, capsules and tablets, and liquid preparations such as syrups and elixirs. Moreover, injections etc. are mentioned as a parenteral administration agent. In addition, when the adiponectin elevating agent and the agent for preventing or ameliorating diseases caused by obesity are used as injections, any of suspensions, emulsions, and liposome preparations may be used.

Each of the above-mentioned preparations can be obtained by, for example, mixing a phospholipid composition with an additive and the like according to a conventional method, for example, the method described in the 15th revised Japanese Pharmacopoeia Preparation General Rules.
Specifically, the granule is prepared by, for example, adding an excipient, a binder, a disintegrant and the like to the phospholipid composition and mixing the mixture uniformly, and then, for example, compression granulation, rolling granulation, spray drying granulation, extrusion Granulation, pulverization granulation, fluidized bed granulation, stirring granulation, or the like may be used. In addition, for example, tablets are produced by directly compression-molding a phospholipid composition with an excipient, a binder, a disintegrant, etc., and mixed evenly, or a tablet is combined with a phospholipid composition and an excipient. Granules produced in advance with an agent, a disintegrant, etc. may be produced as they are, or after adding the above additives and mixing them uniformly, and then compression molding. Capsules are prepared by adding excipients, binders, disintegrants, etc. to a phospholipid composition and mixing them evenly, or if desired, granulated or granulated, and coated with a coating agent. Can be manufactured by filling capsules. Solid oral preparations may contain lubricants, binders, coating agents, colorants, anti-aggregation agents, absorption promoters, solubilizers, stabilizers, vitamins, and the like.
Examples of the excipient include sucrose, lactose, glucose, starch, and mannitol. Examples of the disintegrant include carmellose, starch, crystalline cellulose, and low-substituted hydroxypropylcellulose. Examples of the lubricant include talc, magnesium stearate, calcium stearate or silica. Examples of the binder include gum arabic, carmellose, gelatin, crystalline cellulose, hydroxypropylcellulose, methylcellulose, and popidone. Examples of coating agents include gelatin, sucrose, gum arabic, carnauba wax, etc., or enteric coating agents (for example, cellulose acetate phthalate, methacrylic acid copolymer, hydroxypropyl cellulose phthalate, carboxymethyl ethyl cellulose, etc.). Also good. Examples of the colorant include edible pigments such as edible red No. 2, edible yellow No. 5, and edible blue No. 2. Examples of the stabilizer include anhydrous citric acid, sodium laurate, and glycerol. Examples of vitamins include vitamin A, vitamins B ₁ , B ₂ , B ₆ , B ₁₂ , vitamin C, vitamin D, vitamin E, niacin, pantothenic acid, folic acid, biotin, and the like. Examples of herbal medicine (herbal medicine extract) include licorice, toki and ginger.

  Syrups and elixirs are obtained by suspending or emulsifying a phospholipid composition in a base. Examples of bases that can be used in liquid preparations such as syrups and elixirs include solvents such as purified water, ethanol or a mixture thereof. The liquid preparation further includes a suspending agent (for example, gum arabic, agar, carmellose, hydroxypropyl cellulose, etc.), an emulsifier (for example, polysorbate 80, gum arabic, etc.), and a corrigent (for example, simple syrup, honey, sucrose). , Tartaric acid, etc.), fragrances (eg, methyl salicylate, fennel oil, orange oil, menthol, etc.), preservatives (eg, benzoic acid, sodium benzoate, etc.), buffers (eg, citric acid, hydrogen carbonate, etc.), etc. May be included.

  An injection is produced by dissolving a phospholipid composition and an additive in a solvent according to a conventional method, for example, by an aseptic operation. Examples of the solvent that can be used for the injection include distilled water for injection and physiological saline. The injection may further contain a stabilizer, a solubilizer, a suspending agent, a surfactant, an emulsifier, a soothing agent, a buffer or a preservative. Examples of stabilizers and solubilizers include glutamic acid, aspartic acid, polysorbate 80, and the like. Examples of the suspending agent include cellulose derivatives such as sodium carboxymethylcellulose or methylcellulose, or natural rubbers such as tragacanth or gum arabic. Examples of the surfactant include sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene ether of hydrogenated castor oil, or lecithin. Examples of the emulsifier include polyoxyl stearate, lauromacrogol, polysorbate 80, and gum arabic. Examples of soothing agents include ethyl aminobenzoate, inositol, meprilucaine hydrochloride, lidocaine hydrochloride, chlorobutanol, propylene glycol, or benzyl alcohol. Examples of the buffer include citric acid or a salt thereof, glucose, phosphoric acid or a salt thereof, or acetic acid or a salt thereof. Examples of the preservative include paraoxybenzoic acid ester, benzalkonium chloride, sorbitanate and the like.

  The content of the phospholipid composition in the adiponectin elevating agent of the present invention and the preventive or ameliorating agent for obesity or a disease caused by obesity varies depending on the type of the target disease, but in the case of an oral solid preparation, 20 to 100 % (W / w) may be used. In the case of an oral liquid preparation, it may be about 0.5 to 20% (w / v). In the case of an injection, it may be about 0.1 to 10% (w / v).

Use The phospholipid composition is an active ingredient of an adiponectin elevating agent and an agent for preventing or improving obesity or diseases caused by obesity.

In the present invention, obesity includes “slightly obese” and “obesity” classified by general BMI value and body fat percentage. For example, in adults, the BMI value is 25.0 or higher, the body fat percentage is 20% or higher for men, and 26% or higher for women is classified as obese or slightly obese. In addition, obesity in the present invention includes a state in which various diseases are easily caused by visceral fat obesity such as so-called metabolic syndrome.
Examples of the diseases caused by obesity include lifestyle diseases such as hypertension, hyperlipidemia, diabetes, arteriosclerosis, myocardial infarction, cerebral infarction, angina, stroke, gallstones, gout and the like. Also included are liver disorders caused by obesity (including alcoholic or non-alcoholic liver disorders excluding drug-induced liver disorders). Metabolic syndrome is also included in diseases caused by obesity.

  Further, prevention as used in the present invention includes not only completely suppressing the onset of the disease but also suppressing the progression of the disease, and the treatment includes not only resolving the disease but also improving the disease.

  The dose of the adiponectin-elevating agent of the present invention and the preventive or ameliorating agent for obesity or a disease caused by obesity may be appropriately adjusted depending on the type and degree of the disease. For example, in terms of the amount of the phospholipid composition, About 50 to 2000 mg per person per day is preferable, and about 100 to 1000 mg is more preferable.

  The subject of administration of the adiponectin elevating agent of the present invention and the preventive or ameliorating agent for diseases caused by obesity or obesity are obese people, slightly obese people, metabolic syndrome people, the above-mentioned diseases caused by obesity Healthy individuals including those who are likely to become obese can also be targeted for administration.

Production Method The phospholipid composition described above can be produced, for example, by the method described in JP-A-2007-014270.
Specifically, the adiponectin elevating agent and the agent for preventing or improving obesity or diseases caused by obesity of the present invention include serine and phospholipase D (hereinafter also referred to as “PLD”) in a phospholipid composition containing PC and PE. In the base exchange reaction of phospholipids.
The phospholipid raw material used in this production method may be of any origin, but is preferably of plant origin. Of these, those derived from soybean, rapeseed, sunflower, and palm are preferred. Commercially available products include, for example, SLP-WHITE (PC 25.6% by weight, PE 24.1% by weight, PA 8% by weight, PI 12% by weight, other about 30% by weight), SLP-PI powder (example: PC 18% by weight, PE 22). (% By weight, PA 8% by weight, PI 17% by weight, and other about 35% by weight) (both are manufactured by Sumi Oil).
Moreover, in this manufacturing method, what contains PC and PE in total 35weight% or more should just be used as a phospholipid composition used as a starting material. The plant-derived phospholipid composition and the commercially available product usually contain PC and PE in this ratio. When the content ratio of PC and PE is less than 35% by weight, a high-purity phospholipid such as SLPC55 ( What is necessary is just to adjust to 35 weight% or more by adding (product made from a coconut oil).

  The concentration of phospholipid in the base exchange reaction system is preferably about 5 to 40% by weight, more preferably about 10 to 30% by weight. If it is the said range, sufficient quantity of phospholipid will be obtained and the transfer rate in a base exchange reaction will become practically sufficient.

In this production method, the phospholipid composition is stirred in a two-phase system composed of an organic solvent and water before the base exchange reaction.
The organic solvent is not particularly limited, and aliphatic hydrocarbons such as n-heptane, n-hexane, and petroleum ether; cycloaliphatic hydrocarbons such as cyclopentane and cyclohexane; ethers such as diethyl ether and tetrahydrofuran; methyl acetate And esters such as ethyl acetate; halogenated hydrocarbons such as carbon tetrachloride and chloroform; ketones such as acetone, methyl ethyl ketone, and diethyl ketone. As said organic solvent, what contains a polar solvent and a nonpolar solvent is preferable.

  Examples of the water include ion-exchanged water, purified water, distilled water, tap water, and the like, and acetic acid or the like may be included in these to make a buffer solution for pH adjustment. As the water, a buffer solution is preferably used. The concentration of the buffer is preferably about 0.1 to 2M, more preferably about 0.4 to 1M. In the two-phase system, the mixing ratio of water is preferably 20% by weight or less with respect to the organic solvent, more preferably about 2 to 15% by weight, and still more preferably about 5 to 10% by weight.

  In the above two-phase system, when the organic solvent contains a polar solvent and a nonpolar solvent, the mixing ratio is not particularly limited, but preferably the polar solvent: nonpolar solvent (volume ratio) is 0.5: 9.5. It is about 5: 5. Examples of such a two-phase system composed of an organic solvent and water include a combination of hexane: acetone: water, heptane: acetone: water, hexane: ethyl acetate: water, and the like.

  The stirring of the phospholipid is preferably performed at about 10 to 40 ° C, more preferably about 20 to 30 ° C. The stirring is preferably performed for about 30 minutes to 2 hours. For the stirring, a general stirrer can be used. The strength of stirring is not particularly limited, but may be such that the liquid can be mixed up and down. In the method of the present invention, the phospholipid is usually in a state dissolved in an organic solvent, and the phospholipid, the organic solvent, and water may form an emulsion by the stirring.

In this production method, the phospholipid is stirred in a two-phase system composed of an organic solvent and water, and then a serine-containing compound and PLD are added to perform a base exchange reaction of PC and / or PE.
The concentration of serine in this reaction system is preferably about 20 to 40% by weight, more preferably about 25 to 30% by weight.

  The PLD is not particularly limited as long as it can hydrolyze the phospholipid base moiety, and examples thereof include plant-derived PLD and microorganism-derived PLD. Examples of plant-derived PLD include cabbage and soybean-derived PLD, and examples of microorganism-derived PLD include actinomycetes, filamentous fungus-derived PLD, and the like.

The concentration of PLD in this reaction system is preferably 5 to 200 U, more preferably 20 to 100 U, with respect to 1 g of phospholipid. In addition, 1U uses 95% soybean phosphatidylcholine as a substrate, 37 M 0.2M acetate buffer (pH 4.0, 10 mM CaCl ₂ , 1.3% Triton X-100) 37%. It is the amount of enzyme that liberates 1 μmol of choline per minute when reacted at ° C.

  The base exchange reaction is preferably performed under conditions of about pH 3.5 to 10, more preferably about pH 4 to 9. Moreover, about 10-40 degreeC is preferable and, as for reaction temperature, about 20-30 degreeC is more preferable.

By the base exchange reaction, PC and PE in the phospholipid react with serine to obtain PS and the like. Since PI generally does not easily react with a hydroxyl group-containing compound in the base exchange reaction, PI contained in the raw material phospholipid is contained in the phospholipid composition almost as it is.
Examples of phospholipid compositions obtained by the production method of the present invention include phospholipid compositions containing PI and PA, phospholipid compositions containing PI and PE, in addition to phospholipid compositions containing PI and PS, and the like. It is done.

  After performing the base exchange reaction, for example, the PLD is deactivated by a treatment such as heating, and the organic solvent layer and the aqueous layer are separated by a centrifugal method or the like to obtain the organic solvent layer. Concentrate by removing. Subsequently, crystallization is performed with acetone or ethanol, a solid is obtained by solid-liquid separation, and the phospholipid composition in the present invention can be isolated by drying.

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

Example 1
As a lecithin, 7 g of SLP-PI powder (manufactured by Sakai Oil Co., Ltd.) was mixed and dissolved together with 70 mL of a mixed solution of heptane and acetone to obtain a lecithin solution. Separately, an enzyme-containing serine solution containing 20 g of serine and 500 U of phospholipase D (manufactured by Nagase ChemteX Corporation) in 67 mL of 1 M acetate buffer (pH 4.5) was prepared.
The enzyme-containing serine solution was added to this lecithin solution and stirred at 30 ° C. for 5 hours. Next, 75 mL of a mixed solution of heptane and acetone and 20 g of sodium chloride were added, stirred for 1 hour, and allowed to stand to separate, and then the organic layer was recovered to obtain an organic solvent solution containing PI and PS. This organic solvent solution contained 8 g of solid content.

Example 2
A phospholipid composition was obtained in the same manner as in Example 1 except that the amount of serine and acetate buffer was adjusted to 1.2 times in Example 1.

Example 3
In Example 1, instead of SLP-PI powder (manufactured by Sakai Oil Co., Ltd.) as a raw material lecithin, a mixture of SLP white (manufactured by Sakai Oil Co., Ltd.) and SLP-PC55 (manufactured by Sakai Oil Co., Ltd.) (SLP white) : SLP-PC55 = 9: 1 (weight ratio)) was used in the same manner as in Example 1 to obtain a phospholipid composition.

Example 4
In Example 1, instead of SLP-PI powder (manufactured by Sakai Oil Co., Ltd.) as a raw material lecithin, a mixture of SLP white (manufactured by Sakai Oil Co., Ltd.) and SLP-PC55 (manufactured by Sakai Oil Co., Ltd.) (SLP white) : SLP-PC55 = 8: 2 (weight ratio)) was used in the same manner as in Example 1 to obtain a phospholipid composition.

Example 5
In Example 1, a phospholipid composition was obtained in the same manner as in Example 1 except that the obtained phospholipid composition was further washed with ethanol.
<Composition of phospholipid composition>
The compositions of the phospholipid compositions obtained in Examples 1 to 5 are shown in Table 1 below.

Analysis conditions of the composition The composition of the phospholipid composition was determined by the high performance liquid chromatography method (HPLC method) under the following conditions.
HPLC conditions <br/> Column used: manufactured by GL Sciences Inc. _{Unisil Q NH 2 (4.6mm I.D. ×} 250mm)
Mobile phase: acetonitrile / methanol / 50 mM ammonium dihydrogen phosphate = 1856/874/270
Flow rate: 1.3 mL / min Detection: UV 205 nm

(1) Test 1 on obese model rats
Next, the phospholipid composition of Example 1 (hereinafter referred to as “PIPS”) was given to an obese model Zucker rat, and the effect was verified.
Diet composition and breeding method 6-week-old male Zucker rats were divided into 2 groups (6 animals per group), a control group (TG group) fed with diet according to the AIN-76 composition, and the phospholipid composition of Example 1 A PIPS group to which 2% of (PIPS reinforced soybean phospholipid (trade name: PIPS Nagase) manufactured by Nagase ChemteX) was added was provided. The above two groups were bred for 4 weeks, fasted for 9 hours, and sacrificed by abdominal aorta blood sampling under ether anesthesia, and the liver and adipose tissue were removed. Serum was also obtained from blood.
Table 2 shows the diet composition.

Method for measuring serum parameters Serum lipid concentration was measured by HPLC and enzymatic methods. Serum triglyceride concentration and serum cholesterol concentration are Liposearch (Skylight Biotech, Akita), serum phospholipid concentration is phospholipid E-test Wako (choline oxidase DAOS method, Wako Pure Chemical Industries), serum free fatty acid concentration Was measured using NEFA C-Test Wako (ACS / ACOD method, Wako Pure Chemical Industries). The blood glucose level was measured by the mutarotase / GOD method using glucose CII-Test Wako (Wako Pure Chemical Industries). As liver dysfunction markers, glutamate oxaloacetate aminotransferase (GOT) and glutamate pyruvate aminotransferase (GPT) (transaminase CII-Test Wako, POP / TOOS method, Wako Pure Chemical Industries) were measured. Serum insulin (Levis / Rat Insulin Kit, Shiba Goat) and adiponectin (Mouse / Rat Adiponectin ELISA Kit, Otsuka Pharmaceutical) were measured by ELISA.

Method of measuring liver lipid concentration Total liver lipid is extracted and concentrated by the method of Folch et al. (Folch, J., et al., J. Biol. Chem., 1957; 226: 497-509.). Fletcher method (Flecher, MJ., Clin. Chim. Acta, 1968; 22: 393-397.), Liver phospholipid concentration was determined by the method of Bartlett et al. (Bartlett, GR., J. Biol. Chem., 1959; 234: 466-471.). Liver total cholesterol concentration was measured by cholesterol oxidase DAOS method using cholesterol E-test Wako (Wako Pure Chemical Industries).

Method for Measuring Liver Lipid Metabolism-Related Enzyme Activity Each cell fraction of the liver was prepared as follows. About 2 g of the liver was homogenized with 6 volumes of 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl buffer (pH 7.4) under ice-cooling, and then centrifuged at 700 xg for 10 minutes, and an additional 10,000 xg supernatant was obtained. The mitochondrial fraction was precipitated by centrifugation at 10 min. The supernatant was ultracentrifuged at 125,000 × g for 60 minutes to obtain the cytosol fraction of the supernatant and the precipitated microsomal fraction. The protein amount of each fraction was measured by the Lowry method (Lowry, OH., Et al., J. Biol. Chem., 1951; 193: 265-275.).
The activity of fatty acid synthase (FAS), which is the rate-determining enzyme for fatty acid synthesis, is determined according to the method of Kelly et al. (Kelley, DS., Et al., Biochem. J., 1986; 235: 87-90.). Measurement was performed using The activity of glucose 6-phosphate dehydrogenase (G6PDH) and malate enzyme (ME), where fatty acids supply NADPH to the synthesis system, was determined by the method of Kelly et al. (Kelley, DS., Et al., Biochem. J., 1984). 217: 543-549.) And Ochoa (Ochoa, S., Methods in Enzymology, 1955; 1: 739-753.) And the like, and the measurement was performed using the cytosol fraction. Carnitine palmitoyltransferase (CPT) activity, which is the rate-determining enzyme of fatty acid β-oxidation, is determined by the method of Markwell et al. (Markwell, MA., Et al., J. Biol. Chem., 1973; 248: 3433-3440.) In addition, fatty acid β-oxidation activity in peroxisomes was measured using the mitochondrial fraction according to the method of Lazarow (Lazarow, PB., Methods in Enzymology, 1981; 72: 315-319.). The activity of phosphatidic acid phosphohydrolase (PAP), which is the rate-limiting enzyme in the triglyceride synthesis system, was determined according to the method of Walton et al. (Walton, PA., Et al., Anal. Biochem., 1985; 151: 479-486.) Measurements were made using minutes.

Data obtained by statistical analysis experiments were subjected to a significant difference test using Student's t-test.

result
Effects on body weight, food intake, food efficiency and organ weight
Table 3 shows changes in body weight, food intake, food efficiency, and organ weight in Zucker rats.

  There was no difference between the groups in initial weight, final weight, weight gain and food intake during the 4-week rearing. Regarding organ weight, a significant decrease in liver weight was observed with PIPS intake. In the spleen, a significant decrease was observed with PIPS intake.

Effect on liver lipid concentration
Changes in liver lipid concentration in Zucker rats are shown in Table 4.

  The liver triglyceride and cholesterol levels were significantly reduced by PIPS intake. On the other hand, the phospholipid concentration was not significantly different between the groups.

Effects on serum lipids, blood glucose levels, serum hormones and cytokine levels
Changes in serum lipids in Zucker rats are shown in Table 5.

Table 6 shows changes in triglyceride concentration in Zucker rats.

Table 7 shows changes in cholesterol concentration in Zucker rats.

  A significant increase in serum triglyceride concentration was observed with PIPS intake. A significant decrease in cholesterol concentration was observed with PIPS intake. There were no differences between the groups in phospholipid concentration, free fatty acid concentration, and blood glucose level. Adiponectin concentration was found to increase significantly with PIPS intake. Insulin concentration showed a downward trend with PIPS intake but was not significant.

Effect on liver injury index enzyme activity
The results of the activity of the indicator enzyme for liver damage in Zucker rats are shown in Table 8.

  A significant decrease in liver injury index enzyme activity in the blood was observed in GOT after ingestion of PIPS. GPT also showed a downward trend with PIPS intake, suggesting an improvement in liver function.

Effects on liver triglyceride metabolism-related enzyme activity
Table 9 shows the results of metabolism-related enzyme activities of liver triglycerides in Zucker rats.

  The activities of fatty acid synthase and glucose 6-phosphate dehydrogenase, which are fatty acid synthases, were found to be significantly reduced by ingestion of PIPS. Carnitine palmitoyltransferase activity, which is the rate-determining enzyme of fatty acid β-oxidation system, was found to increase significantly with PIPS intake. There was no difference between the groups in malate enzyme of fatty acid synthesis system, phosphatidic acid phosphohydrase of triglyceride synthesis system and peroxisome β-oxidation activity.

リン脂質組成物の投与により、血清中トリグリセライド濃度、及び総コレステロール(T-Cho)濃度は有意に減少した。
肥満モデルラットに対する試験１の結果及び試験２の結果から、本発明で使用するリン脂質は、高脂血症に対する改善効果、ひいては動脈硬化症に対する改善効果を有することが分る。 (2) Test 2 on obese model rats
In Test 1, the obesity model was prepared in the same manner as in Test 1, except that the period of rat rearing was 3 months instead of 4 weeks, and the number of rats was 6 (n = 6) was changed to 5 (n = 5). The effect of the phospholipid composition on Zucker rats was examined. What was measured was serum triglyceride concentration and total cholesterol (T-Cho) concentration. The measurement results are shown in Table 10 below.

Administration of the phospholipid composition significantly decreased serum triglyceride concentration and total cholesterol (T-Cho) concentration.
From the results of Test 1 and Test 2 for obese model rats, it can be seen that the phospholipids used in the present invention have an improving effect on hyperlipidemia and thus an improving effect on arteriosclerosis.

(3) Test 3 on obese model rats
The effect of the phospholipid composition on the obese model Zucker rat was verified in the same manner as in Test 1, except that the phospholipid composition of Example 5 was used instead of the phospholipid composition of Example 1. Measured were liver weight, liver triglyceride concentration, and various serum parameters. The measurement results are shown in Tables 11 to 13 below.

リン脂質組成物の投与により、肝臓中の中性脂肪濃度が有意に低下し、肝臓重量も低減し、脂肪肝軽減効果が示唆された。
肝機能改善および抗高脂血症効果

リン脂質組成物の投与により、血中AST濃度およびALT濃度の有意な低下が認められ、肝機能の改善が示唆された。また、血中コレステロール濃度の有意な低下及び中性脂肪量の低下傾向が認められた。 Effect on fatty liver

By administration of the phospholipid composition, the neutral fat concentration in the liver was significantly reduced and the liver weight was also reduced, suggesting an effect of reducing fatty liver.
Liver function improvement and antihyperlipidemic effect

The administration of the phospholipid composition significantly decreased the blood AST concentration and ALT concentration, suggesting an improvement in liver function. Moreover, the significant fall of the blood cholesterol level and the fall tendency of a triglyceride amount were recognized.

リン脂質組成物の投与により、血中アディポネクチン濃度の増加傾向が認められた。 Adiponectin increase effect

By administration of the phospholipid composition, an increasing tendency of blood adiponectin concentration was observed.

リン脂質組成物の摂取により、血中AST濃度は変化しなかったが、ALT濃度及びγ−GT濃度が低減し、肝機能改善効果が確認された。 (4) Liver function improvement effect test for human obese
Four men (ages 35-49 years) with a BMI> 25 were given the composition of Example 5 once every morning for 8 consecutive weeks (intake of 400 mg / day). Blood was collected at 2.5 hours after meal, and blood AST concentration, ALT concentration, and γ-GT concentration were measured. Table 14 shows the measurement results.

Ingestion of the phospholipid composition did not change the blood AST concentration, but the ALT concentration and γ-GT concentration were reduced, confirming the liver function improving effect.

リン脂質組成物の摂取により、血液中の総コレステロール濃度の低減が認められた。 (5) Hyperlipidemia improvement test for human obese people
Five men (ages 29-49) with a BMI> 24 were dosed with the composition of Example 5 once every morning for 8 consecutive weeks (intake of 400 mg / day). Blood was collected at 2.5 hours after meal, and blood cholesterol concentration was measured. Table 15 shows the measurement results.

The intake of the phospholipid composition was found to reduce the total cholesterol concentration in the blood.

リン脂質組成物の摂取により、朝の血圧が収縮期で16低下し、拡張期で11低下し、夜の血圧が収縮期で14低下し、拡張期で6低下した。また、ばらついていた高い血圧値が服用約1（朝）〜1.5ヶ月（夜）後に安定した。 (6) Antihypertensive effect test for persons with a history of human hypertension One person (male, 59 years old) with a history of hypertension took the composition of Example 5 once a morning for 8 consecutive weeks (ingestion). Amount 400mg / day). Blood pressure was measured every morning and evening. The results are shown in Table 16, FIG. 1 and FIG.

Ingestion of the phospholipid composition decreased morning blood pressure by 16 in the systolic phase, decreased by 11 in the diastolic phase, decreased by 14 in the systolic phase, and decreased by 6 in the diastolic phase. Moreover, the high blood pressure value which was varied became stable after taking about 1 (morning) to 1.5 months (night).

(7) Adiponectin elevation effect test for humans A granule preparation containing 400 mg of PIPS Nagase (Example 1) in 4 males (age 35-45 years) whose serum adiponectin concentration is low (4-6 μg / mL), It was taken once every morning for 8 consecutive weeks. Blood was collected at 2.5 hours after meal and the serum adiponectin concentration was measured.
The results are shown in FIG. The adiponectin concentration before administration was 4.8 ± 0.7 μg / mL, but increased to 5.7 ± 1.0 μg / mL 8 weeks after administration (p <0.05).

  The adiponectin elevating agent of the present invention and the preventive or ameliorating agent for diseases caused by obesity or obesity promote fat burning in the body by increasing the concentration of adiponectin in the blood, resulting from obesity including obesity and metabolic syndrome It exhibits preventive or ameliorating effects on various lifestyle-related diseases including diseases.

It is a figure which shows transition of the morning blood pressure at the time of making a human with a hypertension history take the phospholipid composition of Example 5. FIG. It is a figure which shows transition of the evening blood pressure at the time of making a human with a hypertension history take the phospholipid composition of Example 5. It is the figure which compared the serum adiponectin density | concentration before and after letting human take the phospholipid composition of Example 5. FIG.

Claims (9)

  An adiponectin elevating agent comprising as an active ingredient a phospholipid composition in which the total ratio of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 50% by weight or more, and the total ratio of phosphatidylserine and phosphatidylinositol is 40% by weight or more.   The adiponectin increasing agent according to claim 1, wherein the ratio of phosphatidylserine to the total of phosphatidylserine, phosphatidylinositol and phosphatidic acid is 30% by weight or more.   The adiponectin elevating agent according to claim 1 or 2, wherein the ratio of phosphatidylinositol to the total of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 40% by weight or less.   The adiponectin elevating agent according to any one of claims 1 to 3, wherein the phospholipid composition contains 20 to 50% by weight of phosphatidylserine and 10 to 25% by weight of phosphatidylinositol based on the whole.   Caused by obesity or obesity, comprising as an active ingredient a phospholipid composition in which the total ratio of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 50% by weight or more, and the total ratio of phosphatidylserine and phosphatidylinositol is 40% by weight or more Preventive or ameliorating disease.   The agent for preventing or improving obesity or a disease caused by obesity according to claim 5, wherein the ratio of phosphatidylserine to the total of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 30% by weight or more.   The agent for preventing or improving obesity or a disease caused by obesity according to claim 5 or 6, wherein the ratio of phosphatidylinositol to the total of phosphatidylserine, phosphatidylinositol, and phosphatidic acid is 40% by weight or less.   The obesity or disease caused by obesity according to any one of claims 5 to 7, wherein the phospholipid composition contains phosphatidylserine in an amount of 20 to 50 wt% and phosphatidylinositol in an amount of 10 to 25 wt% based on the whole. Preventive or ameliorating agent.   The disease caused by obesity or obesity according to any one of claims 5 to 8, wherein the disease caused by obesity is diabetes, hyperlipidemia, hypertension, arteriosclerosis, liver dysfunction, or metabolic syndrome. Preventive or ameliorating agent.

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