JP4359027B2 - Preventive or therapeutic agent for obesity - Google Patents

Description

【００３１】
［実施例２］ＭＣＨ受容体結合阻害試験
ＭＣＨ受容体結合阻害試験は、Jon Chambersらの方法（Nature, 400, 261-265, 1999）を参考にして行った。
【００３２】
すなわち、ヒトＭＣＨ受容体であるＳＬＣ１を発現させたチャイニーズハムスター卵巣細胞（ＣＨＯ細胞）（2〜4μg/タンパク質となるように調整）、0.1nMの［¹²⁵Ｉ］標識した［Phe¹³，Tyr¹⁹］−ＭＣＨ（比活性；2200Ci/mmol、New England Nuclear製）及び最終濃度が500μl/mlになるように調整した製造例２で得られた各プロポリス抽出物とを、22℃で60分間、インキュベートした。その後、グラスファイバーフィルター（Whatman社製、GF/C）を用いて吸引濾過して放射活性をトラップし、次いで、0.3％脱脂粉乳5mlを用いて3回洗浄した。そして、フィルターを乾燥させ、シンチレーション溶液中で一晩浸漬することにより、フィルター中の放射活性を溶出させた後、この溶出物中の放射活性を液体シンチレーションスペクトロメトリー（43％ efficiency）を用いて測定し、以下に示す式を用いてＭＣＨ受容体阻害活性を算出した結果を表２に示した。
【００３３】
また、特異的結合は、0.1μMのＭＣＨと置換される結合と定義し、タンパク質量は、Lowryらの方法（O.H.Lowry, N.J.Rosenbrough, A.L.Farr and R.J.Randall；J. Biol. Chem. 193, 265-275 (1951)）により測定した。
阻害率（％）＝１００−〔（Ｃ_３−Ｂ）／（Ｃ_２ −Ｂ）〕×１００
（式中、Ｃ_３ は、既知量の供試化合物と［¹²⁵Ｉ］標識した［Phe¹³，Tyr¹⁹］−ＭＣＨが共存している状態での［¹²⁵Ｉ］標識した［Phe¹³，Tyr¹⁹］−ＭＣＨの膜画分に対する結合量を表わし、Ｃ_２ は、供試化合物を除いた時の［¹²⁵Ｉ］標識した［Phe¹³，Tyr¹⁹］−ＭＣＨの膜画分に対する結合量を表わし、Ｂは、過剰のＭＣＨ（１×10^-5Ｍ）存在下での［¹²⁵Ｉ］標識した［Phe¹³，Tyr¹⁹］−ＭＣＨの膜画分に対する結合量を表わす。）
【００３４】
なお、本反応系におけるポジティブコントロールとしてのＭＣＨ受容体作動化薬であるＭＣＨのIC₅₀値は、0.25ｎMであった。
【００３５】
表２から、本発明のプロポリス又はその抽出物は、ＭＣＨ受容体に対するＭＣＨの結合を競合的に阻害し、ＭＣＨ受容体と高い親和性を有することが分かる。
【００３６】
【表２】

【００３７】
以下に処方例を示す。
［錠剤の製造］
製造例１で得られたケールのエタノール抽出物を用いて、常法に従って、下記の組成の錠剤を製造した。
（組 成） （配合：質量％）
ケール抽出物 ２４
乳糖 ６３
コーンスターチ １２
グァーガム １
【００３８】
［ジュースの製造］
製造例２で得られたブラジル産プロポリスのエタノール抽出物を用いて、常法に従って、下記の組成のジュースを製造した。
（組 成） （配合：質量％）
冷凍濃縮温州みかん果汁 ５．０
果糖ブドウ糖液糖 １１．０
クエン酸 ０．２
Ｌ−アスコルビン酸 ０．０２
香料 ０．２
色素 ０．１
ブラジル産プロポリス抽出物 ０．２
水 ８３．２８
【００３９】
【発明の効果】
ケール又はその抽出物がＮＰＹ受容体拮抗作用、プロポリス又はその抽出物がＭＣＨ受容体拮抗作用を有することから、これらを含有するＮＰＹ受容体拮抗又はＭＣＨ受容体拮抗剤は、肥満、及びそれらに関連する糖尿病や高脂血症、高血圧などに対する予防又は治療剤、それらを含有する経口用組成物、非経口用組成物、食品又は医薬として有用である。このような組成物は、長期間服用しても安全で、確実な治療を行うことができる。[0001]
[Technical field to which the invention belongs]
The present invention has an antagonistic action on neuropeptide Y receptor or melanin-concentrating hormone receptor, and contains an agent effective for the prevention or / and treatment of obesity or the prevention or / and treatment of obesity-related diseases The present invention relates to an oral composition or parenteral composition, food or medicine.
[0002]
[Prior art]
Obesity is a major problem because it is associated with lifestyle-related diseases. One of the causes of obesity is excessive nutrition such as overeating due to stress and a high fat diet. According to the 1998 National Nutrition Survey by the Ministry of Health, Labor and Welfare, the obese population (over 15 years old) with body mass index (BMI), which is one of the indices of obesity, is estimated to be 23 million by statistics. The average value of BMI is increasing year by year, and the accompanying diseases such as diabetes, hyperlipidemia and hypertension are also increasing rapidly.
[0003]
Mazindol, an appetite-reducing agent used in Japan as a pharmaceutical agent, acts on the appetite regulating center of the hypothalamus and suppresses eating, but is targeted for severe obesity (BMI> 35). However, it has problems of side effects such as psychiatric symptoms. Therefore, it is clinically significant to develop a new type of antifeedant without such problems, and the present invention aims to solve this problem. To date, as related to suppression of feeding, a method using a precursor of serotonin, dopamine, norepinephrine and histamine (Special Table 2000-515139), fruit body of oyster mushroom (Japanese Patent Laid-Open No. 9-20675), avocado (Japanese Patent Laid-Open No. 2002-53474) has been reported. However, they do not describe neuropeptide Y (hereinafter abbreviated as NPY) or melanin-concentrating hormone (hereinafter abbreviated as MCH) involved in feeding behavior and energy metabolism regulation.
[0004]
NPY is a peptide that enhances feeding, is composed of 36 amino acids, and is widely distributed in the central nervous system (Trends Pharmacol. Sci. 12, 111-115, 1991). Subtypes Y1 to Y6 have been reported for the hypothalamic NPY receptor on which NPY acts, and it is known that feeding is suppressed by antagonizing the NPY receptor (Eur. J Pharmacol, 2002, 440 (23), 173-187).
[0005]
MCH is one of the physiologically active substances secreted in the body and is one of the factors that regulate the nerve function, and is known to act as a stimulating factor for appetite in the human body.
[0006]
In recent years, research on MCH has been actively carried out, and by covering this MCH receptor, body weight decreased in mouse experiments. Therefore, compounds that act antagonistically to MCH can prevent obesity or / It has been shown to be effective for treatment (Jon Chambers et al .; Nature, 400, 261-265, 1999).
[0007]
The prophylactic or therapeutic agent for obesity according to the present invention has an NPY receptor or MCH receptor antagonistic action, and is caused by various eating disorders, in particular, enhancement of feeding center or suppression (nonfunction) of satiety center. It is effective in suppressing an abnormal increase in appetite, preventing the accompanying increase in obesity, improving the obesity state, or preventing or / and treating an obesity-related disease. Furthermore, it is promising for the prevention and treatment of overeating observed in various stress-related diseases.
[0008]
[Problems to be solved by the invention]
The object of the present invention is to provide a composition aimed at preventing or treating diseases related to NPY or MCH, such as obesity, and diabetes, hyperlipidemia, hypertension and the like, which are excellent in stability, safety and price. Is to provide things.
[0009]
[Means for Solving the Problems]
As a result of earnest research to solve the above-mentioned problems, the present inventor has NPY receptor antagonistic action, anticancer action and immunity to the kale of a cruciferous plant that is referred to as green juice and drinks the juice. The present inventors have found that propolis used in health supplements for the purpose of activation and the like has an MCH receptor antagonistic action, and completed the present invention.
[0010]
That is, the present invention
Neuropeptide Y receptor antagonist containing ethanol extract of dried kale (excluding treatment and prevention for obesity),
About.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the kale used in the present invention (Brassicca Oleracea L. var. Acephala DC.) Include kitchen kale, mallow kale, bush kale, tree kale, collard, and green leaf kanran. The cruciferous plant is originally a vegetable from southern Europe and is said to be the original species of cabbage. It may be one that is usually used for food, such as leaves, and the cultivation method and cultivation place are not particularly limited.
The propolis used in the present invention is a resinous black lump collected by bees, and may be from any origin, such as Brazilian propolis, Chinese propolis, Australian propolis, Uruguay propolis, Japanese propolis, etc. However, Brazilian propolis and Chinese propolis are preferable from the viewpoint of versatility.
[0012]
The kale or propolis in the present invention distributes a dried product obtained by drying itself, a pulverized product thereof, a supercritical extract, a crude extract with water or an organic solvent such as alcohol, ether, acetone, and the like, and a column. It includes all extract fractions obtained by stepwise purification by various chromatographies such as chromatography. These may be used alone or in combination of two or more.
[0013]
For example, an extract obtained by adding 3 L of 99.5% ethanol extract to 1 kg of propolis ingot from Brazil and soaking overnight at room temperature may be used as it is, or by combining various chromatographies. A purified product may be used.
[0014]
The concentration of kale or propolis extract in the solution of the extracted kale or propolis extract is not particularly limited, but is preferably about 15 to 70% by mass, and preferably about 20 to 60% by mass. If this concentration is less than 15% by mass, it is necessary to evaporate a large amount of a solution such as ethanol or water at the time of drying. If it exceeds 70% by mass, the viscosity of the solution becomes too high, and the processability may be deteriorated.
[0015]
It has not been known at all that the dry matter or extract of kale according to the present invention has NPY receptor antagonism, and the dry matter or extract of propolis has MCH receptor antagonism. This is a new finding.
[0016]
The kale or propolis according to the present invention has excellent NPY receptor antagonism and MCH receptor antagonism, and is intended for the prevention or treatment of obesity and related diabetes, hyperlipidemia, hypertension and the like. It can be used as a prepared food or medicine.
[0017]
The kale of the present invention can be produced as an NPY receptor antagonist, propolis as an MCH receptor antagonist, obesity, and related foods or medicines for preventing or treating diabetes, hyperlipidemia, hypertension, etc. .
[0018]
Examples of the method of applying a medicament such as an NPY receptor antagonist, MCH receptor antagonist, obesity, and related preventive or therapeutic agents for diabetes, hyperlipidemia, hypertension and the like of the present invention include oral administration and non-administration. Any of oral administration can be employed. In administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared by conventional means. Non-toxic pharmaceutical carriers include, for example, glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acids, gelatin, albumin, Water, physiological saline, etc. are mentioned. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.
[0019]
As food, as it is, or in addition to various nutritional components or contained in foods and drinks, NPY receptor antagonist, MCH receptor antagonist, obesity, and diabetes and hyperlipidemia related thereto, It is eaten as a health food or food material useful as a preventive or therapeutic agent for hypertension and the like. For example, after adding the above-mentioned appropriate auxiliaries, it may be used for edible by using conventional means, forming into an edible form, for example, granular, granular, tablet, capsule, paste, etc. It can also be used in various foods such as processed foods such as ham and sausage, processed fish foods such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, fermented milk products, water, fruit juice, milk, You may add and use for drinks, such as tea and a soft drink.
[0020]
The effective dose of the kale of the present invention is appropriately selected and determined according to the patient's age, weight, symptoms, patient grade, administration route, administration schedule, formulation form, strength of the inhibitory activity of the material, etc. In the case of oral administration, the dry weight is usually 0.1 kg / kg body weight or more in terms of an adult, and may be divided into several times a day.
[0021]
The dosage of propolis is generally about 10 to 500 mg / kg body weight per day as dry weight, preferably about 150 to 350 mg / kg body weight per day, and may be divided into several times a day. Good.
[0022]
The toxicity of kale and propolis is low, for example, when ethanol extract of Brazilian propolis was orally administered to rats over a long period of 1000 mg / kg daily for 100 days, no deaths were observed, and body weight changes were also observed. There wasn't.
[0023]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
[0024]
[Production Example 1] Production of kale extract The leaves of kale were dried at 90 ° C to inactivate the enzyme that becomes a bitter taste component of bitterness. Extraction was performed using 20 L of 99.5% ethanol (Wako Pure Chemical Industries, Ltd.) while heating and refluxing 4 kg of the pulverized product with an electric water bath, to obtain 80 g of kale extract.
[Production Example 2] Manufacture of propolis extract Add 3L of 99.5% ethanol (Wako Pure Chemical Industries) to 10g each of Brazilian and Chinese propolis ingots, soak it overnight at 50 ° C, and then remove the ethanol with a rotary evaporator As a result, 324 mg and 216 mg of propolis extract were obtained, respectively.
[0025]
[Example 1] NPY receptor binding inhibition test The NPY receptor binding inhibition test was carried out with reference to the method of M. Munoz et al. (Molecular and Endocrinology, 107, 77-86, 1995).
[0026]
That is, NPY cDNA was inserted into a baculovirus expression vector and expressed in Sf21 insect cells. The membrane preparation is then prepared according to existing methods, and finally buffer A [50 mM Tris (hydroxymethyl) aminomethane, 120 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride. , 0.1% bovine serum albumin (BSA), pH 7.4], and used as a receptor membrane preparation. To this membrane preparation (containing 25 μg protein), [ ¹²⁵ I] peptide YY (final concentration 0.05 nM) and the kale extract obtained in Production Example 1 (final concentration: 500 μg / ml) were added (final solution volume 500 μl). ), Incubated for 2 hours at room temperature, and then centrifuged using the buffer A (excluding BSA) for washing. After performing this three times, the suspension was suspended in buffer B [0.1% sodium dodecyl sulfate (SDS), 2% sodium carbonate, 0.1 N sodium hydroxide], and the receptor-bound radioactivity was measured with a γ counter.
[0027]
Nonspecific binding was measured in the presence of 1 μM NPY. The results are shown in Table 1. The inhibition rate (%) shown here was calculated by the following formula.
Inhibition rate (%) = 100 − [(C ₁ −B ₀ ) / (C ₀ −B ₀ )] × 100
(In the formula, C ₁ represents the amount of binding to the membrane fraction of [ ¹²⁵ I] peptide YY in the presence of a known amount of sample and [ ¹²⁵ I] peptide YY, and C ₀ excludes the sample. It represents a binding amount to the membrane fraction of ^[125 I] peptide YY when the, B ₀ represents an amount of binding membrane fraction ^[125 I] peptide YY in the presence an excess of NPY (1μM).)
[0028]
The IC ₅₀ value (concentration that inhibits NPY receptor binding by 50%) of NPY, which is an NPY receptor agonist, as a positive control in this reaction system was 0.61 nM.
[0029]
From Table 1, it can be seen that the kale of the present invention or an extract thereof competitively inhibits peptide YY binding to the NPY receptor and has a high affinity for the NPY receptor.
[0030]
[Table 1]

[0031]
[Example 2] MCH receptor binding inhibition test The MCH receptor binding inhibition test was performed with reference to the method of Jon Chambers et al. (Nature, 400, 261-265, 1999).
[0032]
That is, Chinese hamster ovary cells (CHO cells) expressing SLC1 which is a human MCH receptor (adjusted to 2-4 μg / protein), 0.1 nM [ ¹²⁵ I] -labeled [Phe ¹³ , Tyr ¹⁹ ] -MCH (specific activity; 2200 Ci / mmol, manufactured by New England Nuclear) and each propolis extract obtained in Preparation Example 2 adjusted to a final concentration of 500 μl / ml were incubated at 22 ° C. for 60 minutes. . Thereafter, the radioactivity was trapped by suction filtration using a glass fiber filter (manufactured by Whatman, GF / C), and then washed three times with 5 ml of 0.3% nonfat dry milk. The filter is dried and immersed in a scintillation solution overnight to elute the radioactivity in the filter, and the radioactivity in the eluate is measured using liquid scintillation spectrometry (43% efficiency). The results of calculating the MCH receptor inhibitory activity using the formula shown below are shown in Table 2.
[0033]
In addition, specific binding is defined as binding that replaces 0.1 μM MCH, and the amount of protein is determined by the method of Lowry et al. (OH Lowry, NJ Rosenbrough, ALFarr and RJ Randall; J. Biol. Chem. 193, 265-275 (1951 )).
Inhibition rate (%) = 100 − [(C ₃ −B) / (C ₂ −B)] × 100
(Wherein, _{C 3} was ^[125 I] labeled with a known amount of test compound [Phe ^13, Tyr ^19] -MCH was ^[125 I] labeled in a state coexisting [Phe ^13, Tyr ¹⁹ ] Represents the amount of MCH bound to the membrane fraction, and C ₂ represents the amount of [ ¹²⁵ I] -labeled [Phe ¹³ , Tyr ¹⁹ ] -MCH bound to the membrane fraction when the test compound was removed. B represents the amount of [ ¹²⁵ I] -labeled [Phe ¹³ , Tyr ¹⁹ ] -MCH bound to the membrane fraction in the presence of excess MCH (1 × 10 ⁻⁵ M).)
[0034]
The IC ₅₀ value of MCH, which is an MCH receptor agonist, as a positive control in this reaction system was 0.25 nM.
[0035]
From Table 2, it can be seen that the propolis of the present invention or an extract thereof competitively inhibits the binding of MCH to the MCH receptor and has a high affinity for the MCH receptor.
[0036]
[Table 2]

[0037]
A prescription example is shown below.
[Manufacture of tablets]
Using the ethanol extract of kale obtained in Production Example 1, tablets having the following composition were produced according to a conventional method.
(Composition) (Composition: Mass%)
Kale extract 24
Lactose 63
Cornstarch 12
Guar gum 1
[0038]
[Manufacture of juice]
Using the Brazilian propolis ethanol extract obtained in Production Example 2, a juice having the following composition was produced according to a conventional method.
(Composition) (Composition: Mass%)
Frozen and concentrated Wenzhou orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Fragrance 0.2
Dye 0.1
Brazilian propolis extract 0.2
Water 83.28
[0039]
【The invention's effect】
Since kale or an extract thereof has NPY receptor antagonistic activity and propolis or an extract thereof has MCH receptor antagonistic activity, NPY receptor antagonists or MCH receptor antagonists containing them are obese and related to them It is useful as a preventive or therapeutic agent for diabetes, hyperlipidemia, hypertension and the like, an oral composition, a parenteral composition, a food or a medicine containing them. Such a composition can be safely and reliably treated even when taken for a long time.

Claims (1)

Neuropeptide Y receptor antagonist containing ethanol extract of dried kale (except for treatment / prevention for obesity).