JP2008163004A - Fat accumulation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a material inhibiting the accumulation of fats in adipocytes of a living tissue, which is easily incorporated in dietary habits and is useful for the prevention and/or amelioration of obesity and to provide a composition in a form industrially utilizing the inhibitor. <P>SOLUTION: Provided are a fat accumulation inhibitor which inhibits the accumulation of fats in adipocytes, comprising as an active ingredient an aqueous constituent of fruits and/or seeds of camellia (Camellia japonica Theaceae) belonging to genus Camellia of family Theaceae and an oral composition containing this, desirably food and drink, for inhibiting the accumulation of fats in adipocytes. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention comprises a fat accumulation inhibitor characterized by containing, as an active ingredient, an aqueous component of a camellia (Camellia japonica) fruit and / or seed belonging to the camellia family Camellia. The present invention relates to an oral composition for suppressing fat accumulation in fat cells.

In recent years, there are an increasing number of obese and obese people in Japan, and obesity has become an important health concern. The reason why obesity is attracting attention is due to the fact that obesity or those who tend to develop it often develop so-called lifestyle-related diseases such as heart disease, arteriosclerosis, hypertension, diabetes, etc., and the mortality due to these diseases is high. It is in. Obesity is known to be a major factor in inducing diabetes, hyperlipidemia, hypertension, fatty liver, and arteriosclerosis. The major causes of death among Japanese are cancer, heart disease, and stroke. However, considering that heart disease and stroke are the main causes of arteriosclerosis, it is understood that there are very many causes of death from arteriosclerotic diseases. it can.

The state of obesity is generally the result of abnormal accumulation of energy sources such as carbohydrates and lipids that are not consumed by various activities such as exercise among the ingested energy, A state in which the body weight has increased beyond the limits of the skeletal system or physiological function. And it becomes clear that the distribution state of the accumulated fat in which the energy source is accumulated as fat in the living body is important in relation to the above-mentioned various diseases. In particular, internal fat type obesity (also called visceral obesity or abdominal obesity) ) Is a combination of two or more of hyperglycemia, hypertension and hyperlipidemia is called metabolic syndrome (metabolic syndrome), and even if each symptom is mild, it is synergistic by overlapping multiple symptoms As the incidence of arteriosclerotic diseases increases, the prevention and treatment are attracting attention as a high-risk group.

In order to prevent obesity, many attempts have been made to prevent obesity through treatment and prevention in the medical field as well as through daily meals and various foods and drinks. For example, L-carnitine or a livestock peptide containing the same as an edible material that aims to regulate metabolism of ingested lipids, promote combustion of body fat, or suppress accumulation (Patent Documents 1, 2, and 3) , Algal extracts (Patent Document 4), carotenoids (Patent Document 5), anthocyanidins (Patent Document 6), green tea catechins (Patent Document 7), porphyran modified low molecular weight compounds (Patent Document 8), etc. Yes.

However, there are few things that can effectively prevent and / or ameliorate obesity symptoms, including practical aspects, and there are few that can be satisfactorily satisfied. Therefore, there has been a strong demand for materials that inhibit the absorption of fat in the diet, suppress the accumulation of fat in the body, or promote the combustion or metabolism of body fat.

The following is known about the camellia described later. That is, camellia has a history of being used as an ornamental horticultural plant since ancient times, and fats and oils collected from seeds are used as fuel oil, hair styling, high-grade edible oil, etc., and xylem is ashed to brew sake. The actual defatted rice bran has been used as a fertilizer for agricultural crops. The defatted cocoons contain saponins and tannins, and there are also proposals for processing them into insecticides (Patent Document 9), agricultural and horticultural nematodes control agents (Patent Document 10), and the like. However, there are no examples of using camellia ingredients for obesity prevention.

JP-A-7-196485 JP-A-10-66515 JP 2000-256200 A JP 2000-72642 A JP 2003-95930 A JP 2002-153240 A JP 2006-131512 A JP 2006-104100 A Japanese Patent No. 170071 JP-A-9-30916

In view of such circumstances, the present invention develops a material that can be easily incorporated into a diet and is useful for preventing and / or improving obesity, and in particular, capable of suppressing fat accumulation in fat cells in vivo. At the same time, an object of the present invention is to provide a composition in an aspect that can be effectively used industrially.

In order to solve the above-mentioned problems, the present inventors have conducted extensive studies on materials for suppressing fat accumulation in adipocytes. As a result, surprisingly, camellia are extremely effective. It has been found that a component capable of suppressing the accumulation of fat in adipose tissue such as, and that it can be effectively used in the fields of foods and drinks, pharmaceuticals, quasi drugs, feeds, etc., and completed the present invention. It came to do.

That is, according to the present invention, there is provided a fat accumulation inhibitor characterized by containing, as an active ingredient, an aqueous component of a camellia (Camellia japonica) fruit and / or seed belonging to the camellia family Camellia. The aqueous component is preferably an extract obtained by extracting a defatted product of Camellia japonica fruits and / or seeds with water and / or a lower alcohol. In addition, the suppression of fat accumulation is desirably to suppress the accumulation of fat in adipose tissue in the living body.

According to the present invention, there is also provided an oral composition for suppressing fat accumulation in mast adipocytes, characterized by comprising the aforementioned fat accumulation inhibitor. The oral composition is preferably a food or drink.

The camellia (Camellia japonica) fruit and / or seed aqueous component according to the present invention is excellent in quality stability and exhibits an effect of suppressing fat accumulation in adipose tissue. Such an effect is remarkably expressed by orally ingesting or administering a fat accumulation inhibitor comprising, as an active ingredient, an aqueous component of camellia (Camellia japonica) fruit and / or seed defatted product according to the present invention. Is done. Accordingly, the fat accumulation-suppressing agent of the present invention is a fat cell in the form of the agent as it is or in a form blended with various conventional products in the field, particularly in the fields of foods and drinks, pharmaceuticals, quasi-drugs, and feeds. It can be effectively used to suppress the accumulation of fat. The fat accumulation inhibitor of the present invention can also be applied to products in the fields of cosmetics and skin external preparations.

The present invention is described in detail below. First, the fat accumulation-suppressing agent of the present invention is a fat accumulation in fat cells in humans and animals, that is, accumulation of fat that is stored as a stored energy source by metabolism of carbohydrates and lipids ingested by meals and the like. It has a function of suppressing cereals, and is characterized by containing, as an active ingredient, an aqueous component of camellia (Camellia japonica) and / or seeds belonging to the genus Camellia of the family Theaceae. .

As the plant belonging to the camellia genus, generally, the camellia camellia belonging to the camellia section, such as Camellia japonica, the tea belonging to the tea section (C. sinensis), the sasanqua belonging to the southern section, C. sasanqua, etc. (C. granthamiana) and the like, and the long-tailed salamander (C. salicifolia) and the like, the Hime Sasanka (C. lutchuensis) and the like belonging to the camellia section are known. Use. Examples of this include C. japonica var. Japonica, C. japonica subsp. Rusticana, C. japonica var. Macrocarpa, C. japonich. Hong Konggenesis, C. reticulata, C. salenensis, Pitaldi var. partardii, and C. partidi var. Yamabe camellia (same kind as Ayaba camellia), wild tea flower (same kind as Ayers camellia), Kushimatsubaki can be mentioned (apple camellia and the like) and the like. What is necessary is just to utilize suitably these camellia which are growing naturally in the Japanese archipelago, the Korean peninsula, the Shandong peninsula of China, etc.

In the present invention, the above camellia nuts and / or seeds are subjected to a compression treatment, a supercritical extraction treatment using a hydrophobic organic solvent such as hexane or heptane, or a liquefied gas such as liquefied carbon dioxide or liquefied propane. Desirably, a defatted product, which is a residue obtained by extracting oil by a method, is used as a raw material for collecting the aqueous component. Here, the camellia seeds and / or seeds may be either early-ripening seeds or mature seeds, and these seeds may be used. From the viewpoint of the yield of the defatted product and the active ingredient, mature seeds or seeds thereof may be used. desirable. More preferably seeds are used. In the present invention, it is preferable to use seeds obtained from mature fruits dried for about 1 to 2 weeks in the sun.

Although the aqueous component which concerns on this invention can be manufactured by arbitrary methods from the camellia fruit, it is preferable to extract from the said defatted material using water and / or a lower alcohol. The lower alcohol has a higher tendency to extract oily components in the defatted product as the carbon number increases, so that a lower alcohol is desirable, such as methanol, ethanol, normal propanol, isopropanol, normal butanol, isobutanol, etc. It can be illustrated. In the case of using a lower alcohol having a large carbon number, it is preferable to increase the water content in order to suppress the extraction of oily components in the defatted product. For example, the water content in the case of propanol is about 20 wt% to about 50 wt%, and the water content in the case of butanol is about 40 wt% to about 70 wt%. Desirable extraction solvents are water, methanol and ethanol, and water-containing alcohols thereof (water content: 0 to 100% by weight).

In order to extract the defatted product, the extraction solvent is added in an amount of about 1 to about 30 times by weight with respect to 1 part by weight of the defatted product, and at about normal temperature or about 1 to 5 atm. After stirring and mixing for 10 minutes to about 3 hours as necessary, the mixture is cooled to room temperature and filtered, and the filtrate is concentrated and dried by an appropriate means such as drying under reduced pressure, spray drying or freeze drying. The dried product may be appropriately pulverized. In this way, a pale yellow to yellow solid which is an aqueous component of camellia nuts and / or seeds according to the present invention can be obtained. The extraction method is preferably performed by repeatedly extracting the extraction residue once extracted, or under a pressure of 1 to 3 atmospheres at about 100 ° C. to about 130 ° C. This increases the yield of the aqueous component according to the present invention. This aqueous component includes saponins, tannins, kaempferol, glycosides thereof and the like.

In the fat accumulation inhibitor of the present invention, the aqueous component as an active ingredient is in a solid, paste, or liquid form, which may be used as it is as a fat accumulation inhibitor. It can also be prepared as a composition by using a conventional method in combination with known additives in applications where an accumulation inhibitor is used. Here, known additives are preferably those commonly used for ingestion, for example, excipients, binders, disintegrants, lubricants, wetting agents, fluidizing agents, preservatives, surfactants. Additives such as stabilizers, diluents, solubilizers, tonicity agents, bactericides, preservatives, flavoring agents, flavoring agents, coloring agents, flavors, etc. can also be used. You may use together the known component which has obesity prevention effects, such as an effect | action, a dietary lipid absorption inhibitory effect, a fat accumulation inhibitory effect, a body fat combustion promotion effect, and its containing material.

In addition to the ingredients described in the above-mentioned patent documents, the ingredients and materials known to have anti-obesity action include green tea catechin, soybean oil unsaponifiable matter, soybean isoflavone, α-lipoic acid, carnitine, coenzyme Q10, phaseolamin, caffeic acid, Dihydroxychalcones, branched amino acids (valine, leucine, isoleucine), anthocyanidins, levan, betaine, ellagitannins, low molecular weight chitosan, rosmarinic acid and its esters, conjugated polyenoic acid, curcumin, resveratrol, forskolin, bergenin, sclerothioline, Diacylglycerol, low molecular weight compounds of porphyran, chlorogenic acid, carotenoids (lycopene, lutein, zeaxanthin, etc.), gymnemic acid, hydroxycitric acid, astilbine, heat-modified gum arabic, hesperidin, molecular weight 5 ~ 150,000 low molecular weight pectin, brewer's yeast produced mannan, uridine, oleanol, oleanolic acid, eugenol, theaflavin, phenyl-OD-glucose, 3,4,6-trimethoxy-OD-glucose, itaconic acid and Salts thereof, hydroxyproline and N-acyl derivatives thereof, conjugates of pregelatinized starch and sucrose fatty acid ester, procyanidins, cyclic and chain polylactic acid having a polymerization degree of 3 to 19, α-methyl-D-xyloside, xylooligosaccharide , L-arabinose, indigestible dextrin, baronelic acid and its glycosides, capsaicin, polyphenol, salacinol, ganoderic acid, soy peptide, whey peptide, beef peptide, bran, rice bran, psyllium seed coat, isolated soy protein, etc. Can do.

Moreover, the dried material of each site | part of the following plant, seaweed, moss, etc., those water, or a lower alcohol extract can be illustrated. That is, guava leaves, gymnema sylvestre leaves, Garcinia cambodia peel, perilla leaves or seeds, evening primrose seeds, quilla bark, eucalyptus leaves, peony flowers, totu leaves, black rice, peanut astringent bark, oolong tea leaves, banana peels, itado stalks, grape seeds Fruit peel, cassis fruit, yellow root leaf, olive leaf, keihi bark, asunaro whole grass, chestnut astringent bark, kumaza leaf, birch bark, amla fruit, mulberry leaf, chickpea, red pepper root, coleus forskori root, kawara ketsumei leaf or saya, Turmeric Root, Seaweed, Clove Mushroom, Oregano Whole Grass, Akamegashi Bark, Vulcania Bark Bark, Hypericum Whole Grass, Salacia Reticulata Root, Shrimp Grass, Aloe Mesophyll, Ezoukogi Root, Ganoderma maitake, Enokitake, Kabanoanatake, Agaricus Mushroom, etc. Entity and fungus Body, defatted rice germ, a kale leaves and the like. In addition, the fat accumulation inhibitor of this invention is not restrict | limited at all by these illustrations.

When the fat accumulation inhibitor of the present invention is used as a composition, it can be made into oral preparations such as granules, tablets, capsules, and liquids. The content of the aqueous component in such a pharmaceutical composition is difficult to define uniformly depending on the type and content of the combined raw material, but is generally about 0.01% to 90% by weight, more preferably about 0.1% by weight. To about 70% by weight. When the content is less than about 0.01% by weight, the desired effect of the present invention is not recognized, and when it exceeds about 90% by weight, it is difficult to prepare a practical pharmaceutical composition. The fat accumulation-suppressing agent of the present invention is preferably used in a mode of being taken or administered orally. A suitable amount of the fat accumulation inhibitor of the present invention when orally ingested or administered is about 10 mg to about 1,000 mg, preferably about 30 mg per day for a human adult, based on the aqueous component contained in the agent. To about 500 mg, more desirably from about 50 mg to about 300 mg.

A major feature of the usefulness of the fat accumulation-inhibiting agent of the present invention is that it inhibits the accumulation of fat in fat cells of living tissues as well as preventing and promoting the increase in body weight, and in addition to this, fat that is ingested with meals It is to inhibit the absorption of or to burn or metabolize body fat. Therefore, the fat accumulation-inhibiting agent of the present invention is applied for the prevention and / or improvement of obesity symptoms based on prevention of weight gain and promotion of reduction, which is one of the obesity evaluation indexes, and the accumulation of fat in fat cells. It is more suitably applied for suppression, inhibition of dietary fat absorption, and / or prevention and / or improvement of obesity symptoms based on the promotion of combustion or metabolism of body fat such as visceral fat and subcutaneous fat in the living body.

In the present invention, the aforementioned fat accumulation inhibitor can be used as it is in various forms of foods, beverages, pharmaceuticals, quasi-drugs, feeds, and other industrial fields. It can utilize also in the aspect used as a part. In particular, an oral composition for suppressing the accumulation of fat in fat cells is preferred, and the most preferred embodiment of this oral composition is a food or drink. This example will be described below, but the present invention is not limited thereby.

Specific examples of food and drink include beverages such as vegetable juice, fruit juice drinks, soft drinks, tea, soup, jelly, pudding, yogurt, cake premix products, confectionery, sprinkles, miso, soy sauce, sauce, dressing, mayonnaise, Vegetable cream, seasonings for grilled meat sauce and noodle soup, noodles, udon, soba noodles, spaghetti, processed meat and fish products such as ham and sausage, hamburger, croquette, sprinkle, boiled, jam, milk, cream, butter, spread In addition to various processed foods such as powdered, solid and liquid dairy products such as cheese and cheese, margarine, bread, cakes, cookies, chocolate, candy, gummi, gum, etc., powdered, granular, pills, tablets , Soft capsule, hard capsule, paste or liquid dietary supplement, food for specified health use, functional food , Mention may be made of health food, diet, etc. of the concentrated liquid diet and dysphagia for food.

In addition, in the food / beverage products of this invention, this contains the aqueous component of the camellia (Camellia japonica) fruit and / or seed which belong to the camellia genus (Camelia) of the Camellia family (Theaceae) as an active ingredient, It can be set as the aspect which attached | subjected the indication of at least 1 among the things for the accumulation | storage suppression of fat in the fat cell.

In order to produce these foods and drinks, the fat accumulation inhibitor of the present invention and known raw materials may be used, or a part of the known raw materials may be replaced with the aforementioned fat accumulation inhibitor and produced by conventional methods. For example, the fat accumulation inhibitor of the present invention can be added to excipients such as glucose (dextrose), dextrin, lactose, starch or processed product thereof, cellulose powder, vitamins, minerals, fats and oils of animals, plants and fish, White (proteins derived from plants and animals, hydrolysates thereof, etc.), carbohydrates, pigments, fragrances, antioxidants, surfactants, other edible additives, powders and extracts containing various nutritional functional ingredients, etc. Mixed with raw materials and processed into powders, granules, pellets, tablets, etc., processed into general processed foods of the above examples by conventional methods, mixed liquids such as gelatin, sodium alginate, carboxymethyl cellulose, etc. It is suitable to use as a dietary supplement or health food by coating it with the above coating agent and forming it into a capsule, or processing it into a beverage (drink) form.In particular, tablets, capsules and drinks are desirable.

The ratio of the fat accumulation inhibitor of the present invention to be blended in such a food or drink is the form of the food or drink, the aqueous component in the fat accumulation inhibitor of the present invention (aqueous component of camellia camellia and / or seed). The content of the aqueous component in foods and beverages is more preferably about 0.01% to about 90% by weight, although it is difficult to specify uniformly depending on the content of other ingredients, the types and ingredients of the ingredients, and the amount of ingredients. The formulation is designed by appropriately combining the fat accumulation inhibitor of the present invention with other known ingredients for the production of food and drink so that it is about 1% by weight to about 50% by weight. What is necessary is just to prepare. In foods and beverages in which the content of the aqueous component is less than about 0.01% by weight, a large amount of the food and beverages must be ingested in order to expect the desired effect of the aqueous component, while the amount of the aqueous component If it exceeds about 90% by weight, it may be difficult to produce a practical food or drink. In the case of a human adult, the food / beverage product of the present invention can be arbitrarily selected so that the daily intake of the aqueous component is about 10 mg to about 1,000 mg, desirably about 30 mg to about 500 mg, more desirably about 50 mg to about 300 mg. This method can be taken into the body at the same time as, for example, or before or after meal intake, by oral intake, tube administration, or the like.

EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited at all by this. In each example, all percentages, parts and ratios are by weight unless otherwise indicated.

Production Example 1
After coarsely crushing dried seeds of C. japonica var. Japonica from Goto, Nagasaki Prefecture, steaming and then pressing to obtain a press knead separated from the compressed oil, followed by adding normal hexane to the press koji and extracting by a conventional method After processing, the extract was separated and the extract was collected. The extract was washed with normal hexane to remove the oil, and a defatted material was collected. Water (300 mL) was added to 100 g of the defatted material, heated to 85 ° C. under normal pressure and stirred appropriately for 1 hour, cooled to room temperature, and filtered to separate the filtrate. 200 mL of water was again added to the filtration residue and heated in the same manner. After cooling, the filtrate was collected by filtration. Both filtrates were combined, concentrated under reduced pressure, freeze-dried and pulverized to obtain 15.7 g of a powder (referred to as sample 1) containing an aqueous component according to the present invention. When this powder was hydrolyzed and analyzed by HPLC, it contained 16.1% sapogenin which is an aglycon of saponin and 2.0% kaempferol which is a kind of flavonol.

Production Example 2
The dried seeds of Yakushima-made Yakushima pine (C. japonica var. Macrocarpa) were defatted by the method described in Production Example 1 and the defatted material was collected. 300 mL of water was added to 100 g of this defatted material, heated at 120 ° C. under a pressure of 2 atm for 25 minutes, cooled to room temperature, and filtered to separate the filtrate. 200 mL of water was again added to the filtration residue and heated in the same manner. After cooling, the filtrate was collected by filtration. Both filtrates were combined, concentrated under reduced pressure, freeze-dried and pulverized to obtain 16.7 g of a powder (referred to as sample 2) containing an aqueous component according to the present invention. The powder was hydrolyzed in the same manner as in Production Example 1 and analyzed by HPLC. As a result, the sapogenin content was 14.8% and the kaempferol content was 2.3%.

Production Example 3
250 g of water-containing ethanol (water content 50%) was added to 100 g of the defatted product obtained by the method described in Production Example 1, and the mixture was heated to reflux at 80 ° C. for 1 hour, cooled to room temperature, and filtered to separate the filtrate. To this filtration residue, 200 mL of hydrous ethanol (water content 50%) was added again and heated in the same manner. After cooling, the filtrate was collected by filtration. Both filtrates were combined, concentrated under reduced pressure, lyophilized and pulverized to obtain 11.9 g of a powder containing the aqueous component according to the present invention (referred to as sample 3). The powder was hydrolyzed in the same manner as in Production Example 1 and analyzed by HPLC. As a result, the sapogenin content was 12.5% and the kaempferol content was 2.5%.

Production Example 4
Ethanol (purity 99.5%) 200 mL was added to 100 g of the defatted product obtained by the method described in Production Example 2, and the mixture was heated to reflux at 80 ° C. for 1 hour, cooled to room temperature, and filtered to separate the filtrate. 200 mL of ethanol (purity 99.5%) was again added to the filtration residue and heated in the same manner. After cooling, the filtrate was collected by filtration. Both filtrates were combined, concentrated under reduced pressure, lyophilized and pulverized to obtain 4.8 g of a powder (referred to as sample 4) containing an aqueous component according to the present invention. The powder was hydrolyzed in the same manner as in Production Example 1 and analyzed by HPLC. As a result, the sapogenin content was 14.3% and the kaempferol content was 2.8%.

Production Example 5
In Production Example 1, except that the dried seed was replaced with immature fruit (whole seed-containing seed), the same treatment was performed to obtain a defatted koji, and then 13.4 g of a powder containing an aqueous component (referred to as sample 5). Got. The powder was hydrolyzed in the same manner as in Production Example 1 and analyzed by HPLC. As a result, the sapogenin content was 13.8% and the kaempferol content was 2.2%.

Test Example 1: Inhibition of fat accumulation in fat cells The effect of the fat accumulation inhibitor of the present invention (Sample 1 to Sample 5) on the fat accumulation action of adipocytes was examined by the method described below. That is, using a Petri dish (φ10 cm), 3T3-L1 cells (mouse fetal-derived obese preadipocytes: 3T3-L1 manufactured by Dainippon Sumitomo Pharma Co., Ltd., hereinafter simply referred to as cells) were treated with 10% fetal bovine serum ( 1.4 × 10 ⁵ cells were seeded on a D-MEM medium (manufactured by Gibco) (Sigma, high glucose) and cultured for 3 days until it became subconfluent (approximately 80% density). Subsequently, the medium was removed, and the cells were washed twice with 5 mL of PBS, and further with 5 mL of 0.02% EDTA (Sigma, reagent) solution, and then with a 0.25% trypsin solution (manufactured by Nacalai Tesque) Reagents) Cells were collected using 5 mL, centrifuged (4 ° C., 1,000 rpm, 5 minutes) to remove the supernatant, and washed twice with PBS to obtain cells. The cells were repeatedly cultured under the above conditions and subcultured for 10 generations.

Using a 24-well 2 cm ² cell culture plate (Asahi Techno Glass Co., Ltd.), the subcultured cells were added to 1.5 × 10 ⁵ cells / cm ^{2 in} the 10% fetal bovine serum-added D-MEM medium. And then cultured for 2 days. Next, the medium was removed, 10% fetal bovine serum (Daiichi Chemicals Co., Ltd., reagent), 0.5 mM 1-methyl-3-isobutylxanthine (Sigma, reagent), 0.25 μM dexamethasone (Sigma) And D-MEM medium containing 0.1 μM troglitazone (manufactured by Sigma, reagent) and 5 μg / mL insulin (manufactured by Sigma, reagent) were stimulated to differentiate into adipocytes. After culturing this for 3 days, the medium is removed and cultured in D-MEM medium containing 10% fetal calf serum and 5 μg / mL insulin to which the above sample has been added to a final concentration of 10, 30 or 100 μg / mL. Subsequently, the medium was replaced every 2 days for 6 days. After completion of the culture, the medium was removed, and the cells were washed 3 times with 1 mL of PBS to obtain cultured adipocytes. In the above method, the D-MEM medium was added with penicillin (final concentration 100 IU / mL) and streptomycin (final concentration 0.1 mg / mL), and all cell cultures were CO ₂ incubator (37 ° C., 5% CO 2). ₂ under enhanced gas phase). In order to quantify the triglyceride content and protein content of the cultured adipocytes thus obtained, 1% (w / v) Triton (registered trademark) X-100 (manufactured by Kishida Chemical Co., Ltd., reagent) 200 μL After leaving for 15 minutes, the cells were disrupted by pipetting, and this was used as a sample for each quantitative measurement.

Triglyceride was quantified by the acetylacetone method. That is, after 3 mL of isopropanol was added to 0.1 mL of the sample and mixed, 0.4 g of activated alumina (manufactured by Wako Pure Chemical Industries, Ltd., for column chromatography, about 300 mesh) was added and mixed three times every 5 minutes. did. Thereafter, 2 drops of 5% (w / v) aqueous potassium hydroxide solution was added to 1 mL of the centrifuged supernatant (2,000 rpm, 5 minutes) with a Pasteur pipette, mixed and heated at 37 ° C. for 15 minutes. Next, 1 mL of ammonium acetate buffer solution (2.4 M ammonium acetate, 11% isopropanol, pH 5.5) was added thereto and mixed, and then 0.1 mL of 50 mM sodium metaperiodate aqueous solution was added and mixed, and then at room temperature. Let stand for 15 minutes. A coloring solution (97 mM acetylacetone, 65% isopropanol) (2 mL) was added to this and mixed, followed by heating at 37 ° C. for 40 minutes. Thereafter, the reaction was stopped by cooling with running water for 3 minutes, and the absorbance at 410 nm was measured. The triglyceride content in the cultured cells was calculated from a calibration curve prepared using a glycerin standard solution (containing glycerin 20.8 mg / dL corresponding to a predetermined triolein concentration, isopropanol 8%).

The protein was quantified using a BCA protein assay kit (PIERCE). Since the amount of protein in the cells is known to correlate with the number of cells, the triglyceride content accumulated in the cultured adipocytes was determined based on the amount of protein in the cells.

In order to observe the state of fat accumulated in the cultured cells, the cells were cultured separately under the above culture conditions, the medium was removed, the cells were washed with 1 mL of PBS, and then 10% neutral formalin solution (manufactured by Kishida Chemical Co., Ltd.). , Reagent) was fixed with 500 μL for 2 hours. Next, the formalin solution was removed, the cells were washed with water, and an oil red O solution (oil red O (manufactured by Sigma, reagent) dissolved in isopropanol to a concentration of 0.35% (w / v) and pure water Were mixed at a ratio of 6: 4, and allowed to stand for 10 minutes, and then filtered through a filter). Thereafter, the oil red O solution was removed, washed with water, and examined under a microscope.

The results are shown in Table 1. In the table, the numerical values are shown as relative values when the value of the control test (when no sample is added) carried out at the same time is taken as 100. From the data in Table 1, the aqueous component of the defatted product according to the present invention has an action of suppressing the accumulation of fat in adipocytes, and this action can be achieved by heating the defatted product under normal pressure or under pressure. In the case of extraction, it was confirmed that it became remarkable when the aqueous ethanol was extracted under heating. This was supported by the aforementioned microscopic observation results of the fat-stained specimen.

Reference Test Example 1: Dietary Fat Absorption Inhibitory Effect A 3-week-old ICR female mouse (purchased from Nippon Claire Co., Ltd.) is fed with a food (Japan Claire Co., Ltd.) under a temperature and humidity control in a 12-hour light-dark cycle. Made by CE, CE-2) and drinking water ad libitum for 1 week. The total average body weight (g) at this time point was 16.1 ± 1.8 (standard error). Thereafter, a high-fat diet was freely ingested for 8 weeks as a group of 14 animals, sacrificed under ether anesthesia by a conventional method, body weight was measured, and the amounts of neutral lipids in plasma and liver tissue were measured. Here, the composition of the high fat diet of the control group is beef tallow: 40 parts, casein: 36 parts, corn starch: 10 parts, sugar: 9 parts, mineral mixture (Oriental Yeast Co., Ltd., AIN-76): 4 parts , And a vitamin mixture (Oriental Yeast Co., Ltd., AIN 1977): 1 part, and in the case of the sample addition group, a part of the corn starch of the high fat diet is a sample (sample 1, sample 3, sample 4 or sample 5) The added amount was 5%.). The neutral lipid content in the liver was determined by homogenizing a part of the extracted liver tissue (0.5 g) in 4.5 mL of Krebs Ringer's phosphate buffer (pH 7.4), and adding 0.2 mL of the homogenate to chloroform. After extraction with 4 mL of methanol / methanol (2/1: volume ratio), the extract was obtained by concentration and desolvation in a nitrogen atmosphere, and this was used as a measurement sample. Triglyceride E test kit (manufactured by Wako Pure Chemical Industries, Ltd.) ). The neutral lipid content in plasma was also measured in the same manner.

The results are shown in Table 2. In the table, the numerical value represents an average value ± standard error (n = 14, ANOVA statistical processing). There was no significant difference in the amount of energy consumed between the subject group and the sample addition group in this study. From the data in Table 2, it is clear that the weight gain is significantly less in the sample-added group than in the control group ingested the high-fat diet, and the triglyceride content in liver tissue and plasma is significantly reduced. It was. This suggests that the intake of the sample according to the present invention suppresses the absorption of fat derived from meals and promotes the metabolism (combustion) of body fat, and the results of this test will be described later. Considering the result of Test Example 2 together, it was determined that the former contribution was particularly large.

Reference Test Example 2: Combustion promoting action of body fat A 3-week-old ICR female mouse (purchased from Nippon Claire Co., Ltd.) was fed under the control of temperature and humidity in a 12-hour light / dark cycle (made by Nippon Claire Co., Ltd.) CE-2) and drinking water were freely ingested and pre-bred for 1 week, and then the high fat diet described in Reference Test Example 1 was freely ingested and reared for 8 weeks. The total average body weight (g) after the preliminary breeding and after the high fat diet was 16.3 ± 1.8 (standard error) and 38.5 ± 3.2 (standard error), respectively. Thereafter, as a group of 14 animals, a low-fat diet was freely ingested over 4 weeks, sacrificed under ether anesthesia by a conventional method, body weight was measured, and the amounts of neutral lipids in plasma and liver tissue were measured. Here, the composition of the low-fat diet of the control group is corn starch: 65 parts, casein: 25 parts, refined soybean oil: 5 parts, cellulose: 5 parts, mineral mixture (Oriental Yeast Co., Ltd., AIN-76): 4 parts and a vitamin mixture (Oriental Yeast Co., Ltd., AIN 1977): 1 part, and in the case of the sample addition group, a part of the corn starch of the low-fat diet sample (sample 1, sample 3, sample 4 or sample) 5 and the addition amount was 5%.) Each neutral lipid content in the liver and plasma was measured by the method described in Reference Test Example 1.

The results are shown in Table 3. In the table, the numerical value represents an average value ± standard error (n = 14, ANOVA statistical processing). There was no significant difference in the amount of energy consumed between the subject group and the sample addition group in this study. From the data in Table 3, it is clear that the weight loss was significantly higher in the sample-added group and the triglyceride content in the liver tissue and plasma was significantly reduced compared to the control group ingested the low-fat diet. It was. This suggests that ingestion of the sample according to the present invention suppresses the absorption of diet-derived fat and promotes the metabolism (burning) of body fat. Considering the result of Test Example 1 together, it was judged that the latter contribution was particularly large.

Prototype example 1
Any one of Samples 1 to 5 as the fat accumulation inhibitor of the present invention was subjected to a capsule filling machine, and a gelatin-coated hard capsule formulation having an internal volume of 175 mg per grain was prepared by a conventional method. The other samples were processed in the same manner to produce five types of gelatin-coated hard capsule formulations. These capsule preparations can be used as dietary supplements, medicines and the like that can be taken orally.

Prototype example 2
Sample 3 as a fat accumulation inhibitor of the present invention: 30 parts, guava leaf extract (manufactured by BN Co., Ltd.): 0.5 part, α-lipoic acid (manufactured by Cargill): 5 parts, corn starch (Japanese corn starch ( 100 parts, tricalcium phosphate (made by Yoneyama Chemical Co., Ltd.): 50 parts and riboflavin (made by DSM Nutrition Japan Co., Ltd.): 7 parts were charged into a mixer and mixed with stirring for 10 minutes. . This mixture was subjected to a direct compression tableting machine to prepare uncoated tablets having a diameter of 7 mm, a height of 4 mm, and a weight of 150 mg / piece, and then a shellac film was formed by the coating machine to produce a tablet-shaped food.

Prototype example 3
10 g of sample 4 as a fat accumulation inhibitor of the present invention was added to 1 L of commercially available grapefruit juice and mixed well to produce a homogeneous grape flavored drink. Even when stored in the refrigerator for 3 weeks, the appearance and flavor were neither abnormal nor uncomfortable. It should be noted that this product can be marked with an indication that it is intended to suppress fat accumulation in fat cells of living tissue.

The fat accumulation inhibitor comprising, as an active ingredient, an aqueous component of camellia (Camellia japonica) berries and / or seeds belonging to the genus Camellia belonging to the Camellia family of the present invention is a fat cell of living tissue by ingesting this Therefore, it can be effectively used for foods and drinks, pharmaceuticals, quasi-drugs, feeds and the like for preventing and / or improving obesity symptoms.

Claims (5)

  A fat accumulation inhibitor comprising, as an active ingredient, an aqueous component of fruits and / or seeds of Camellia japonica belonging to the genus Camellia.   2. The fat accumulation suppression according to claim 1, wherein the aqueous component is an extract obtained by extracting a defatted product of Camellia japonica fruits and / or seeds with water and / or a lower alcohol, belonging to the genus Camellia. Agent.   The fat accumulation inhibitor according to claim 1 or 2, wherein the fat accumulation suppression is suppression of fat accumulation in fat cells. An oral composition for suppressing fat accumulation in adipocytes, comprising the fat accumulation inhibitor according to any one of claims 1 to 3. The oral composition for suppressing fat accumulation in fat cells according to claim 4, wherein the oral composition is a food or drink.