JP6513404B2 - Fat burning promoter and hypothermia improving agent - Google Patents

Description

The present invention relates to a fat combustion promoter and a hypothermia improving agent characterized by containing passionflower extract.

Adipose tissue is one of the important organs present in the living body, and is roughly divided into two types, white adipose tissue and brown adipose tissue. White adipose tissue is mainly composed of white adipocytes that store energy as neutral fat in cells and has a function to store surplus energy in the living body. In addition, when the living body needs energy, such as during exercise or starvation, these cells break down the stored neutral fat into free fatty acids, and via adenosine's triphosphate via the electron transport system present in mitochondria. By synthesizing ATP, it plays a role of supplying energy to the living body.

On the other hand, brown adipose tissue is mainly composed of brown adipocytes. Like white fat cells, neutral fat is stored in cells, but due to the function of uncoupling protein 1 (UCP1), neutral fat is transferred to heat without undergoing ATP synthesis in the electron transfer system. Convert. This heat conversion plays a role of regulating the amount of neutral fat in the living body by consuming the energy which has been ingested in excess (Non-patent documents 1 to 3). Moreover, as a difference from white fat cells, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1 alpha: peroxisome proliferator-activated receptor gamma coactivator 1-alpha), cell death activator CIDE-A (CIDEA: cell) It is known that death activator CIDE-A) and pyruvate dehydrogenase isozyme 4 (PDK4: pyruvate dehydrogenase kinase isozyme 4) are specifically expressed (Non-patent Document 4).

Although the balance of neutral fat content in the living body is maintained by the function of these white fat cells and brown fat cells, excess intake of energy causes excess fat accumulation in white fat tissue, resulting in obesity This is considered to be the main cause of the onset of the metabolic syndrome (Non-patent Documents 5 and 6).

It is necessary to consume energy efficiently to improve obesity and metabolic syndrome. Therefore, research focusing on brown adipocytes that consume energy is increasing year by year, and the weight gain of brown adipose tissue by genipa oil and conjugated linoleic acid (patent documents 1 and 2), UCP 1 in brown adipocytes by components containing wasabi wasp The upregulation of expression (Patent Document 3) and the like are known.

On the other hand, as a result of recent researches, white fat cells have the property of expressing UCP1 similar to brown fat cells by cold stimulation, response via β3 adrenergic receptor, or iricin which is a hormone induced by exercise. It has been confirmed that they become cells (Non-patent Documents 7 and 8). Therefore, it has not only the effect of enhancing brown adipocytes and the expression of UCP1 but also the effect of consuming white fat neutral fat as energy for improving obesity and metabolic syndrome. If you can find out, you can expect higher effects.

The body temperature of a homeothermic animal such as a human being is kept within a certain range by homeostasis. Body temperature is roughly divided into two in relation to the environment, the temperature at the deep part of the body that is not easily influenced by the environmental temperature is called the core body temperature, and the temperature of the susceptible surface is called the body surface temperature. Unlike body surface temperature, core temperature is constantly regulated by thermoregulation, and generally, rectum temperature, oral temperature, axillary temperature and tympanic temperature are measured with a thermometer. On the other hand, the body surface temperature is the temperature of the outer layer of the body, and changes with the environmental temperature. Body surface temperature is measured using thermography or the like.

Due to irregular living and excessive stress, the decrease in basal metabolic rate and the influence of low temperature environment lead to hypothermia with decreased core body temperature. Hypothermia causes various physical functions such as chills, discomfort, decreased immunity, and cold. Methods to improve blood flow such as ginseng and ginger, methods to promote blood circulation by massage, and methods such as intake of warm food and bathing have been proposed as methods to improve hypothermia, but those methods are body surface Since it only raises the temperature transiently and can not raise core body temperature, it has not resulted in the improvement of the fundamental hypothermia (patent documents 4 and 5).

One of the roles of brown adipocytes is maintenance of body temperature, which mainly works during hibernation of animals and newborns to produce heat without shaking their bodies. On the other hand, it has been confirmed that core body temperature rises with the increase of UCP1 expression, and brown adipocytes are activated at the time of recovery when hypothermia is caused by low temperature exposure or anesthesia etc. (Non-patent document 9) ~ 11). Therefore, it can be said that acting on brown adipocytes to increase heat production is effective in improving hypothermia.

The composition (patent document 6) which raises the sympathetic nerve activity of brown adipose tissue for the purpose of a rise in core body temperature is examined. However, the prior art only increases UCP1 which contributes to thermogenesis in brown adipocytes. Even in white fat cells as proposed in the present invention, a higher hypothermia improvement effect can be expected by increasing UCP1.

Passionflower is a plant of the family Passifloraceae native to Brazil, and its scientific name is called Passiflora incarnata (Passiflora incarnata), and it is mainly used as herbs that it is of the incarnata species (Cabort diatoma), edulis species (Buda monotonicho) The main components of leaves, branches and stems are flavonoids including polysaccharides and alkanoids. As the main medicinal effects of passionflower, sedation, mental stability, glycation inhibition and the like are known (Patent Documents 7 and 8). In addition, differentiation promoting action from stem cells to brown adipocytes is also known (Patent Document 9), but the increase in brown adipose tissue weight in vivo and UCP1 expression enhancing action in white adipocytes are not known yet.

Patent No. 3951271 gazette Patent No. 3207823 Unexamined-Japanese-Patent No. 2006-328056 Japanese Patent Application Publication No. 2003-40788 JP, 2007-145773, A JP, 2014-15430, A JP 2002-101850 A Patent Document 1: Japanese Patent Application Publication No. 2008-088102 JP, 2013-151437, A Shizuka Akira, History of Medicine, 1998, 184, 513-517 Saito, Masayuki, Science of Obesity, The 124th Japan Medical Association Symposium, 62-70 Yoshio Kagawa, History of Medicine, 1998, 184, 529-533 Jun, Wu. , Et al. , Cell, 2012, 150, 366-376. Shigeru Aoki et al., Japan Clinical Clinic, 2006, volume 64, special issue 9, 175-179 Akihiro Ogawa et al., Experimental Medicine, 2007, 25, Special Issue 15, 54-60 Irie Yukiko et al., Obesity Research, 2007, 7, 1, 63-65 Bostrom, P .; , Et al. , Nature, 2012, 481, 463-468 Kengo, Azushima. , Et al. , PLOS ONE, 2013, 8, 10 Bin, Feng. , Et al. , Ann. N. Y. Acad. Sci, 2013, 1281, 160-177 Shimizu, Y. , Et al. , Am. J. Physiol, 2013, 1281, 160-177

It is an object of the present invention to provide a routinely available and highly safe fat burning promoter that can improve obesity, metabolic syndrome and hypothermia by promoting fat burning in brown fat cells and white fat cells. And providing a hypothermia improving agent.

As a result of intensive studies to solve the above problems, the present inventors have found that passionflower extract promotes differentiation into brown adipocytes, increased weight of brown adipose tissue, and enhanced UCP1 expression in brown adipocytes and white adipocytes. The inventors have found that they exhibit a fat combustion promoting effect and a hypothermia improving effect, and have completed the present invention.

The passionflower extract used in the present invention is obtained by extraction with a solvent from passionflower leaves, anthers, stems, stems, fruits and flowers, but it is preferable to use whole grass. The extraction method is not particularly limited, and it may be heat extraction or normal temperature extraction.

As a solvent used for extraction of passion flower, for example, water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohol (1,3-butylene, etc.) Glycol, propylene glycol, glycerin etc., ketones (acetone, methyl ethyl ketone etc.), acetonitrile, esters (ethyl acetate, butyl acetate etc.), hydrocarbons (hexane, heptane etc.), ethers (ethyl ether, tetrahydrofuran, propyl) Ether, polyoxyethylene cetyl ether and the like) can be mentioned, preferably polar solvents such as water and lower alcohols, and more preferably water, ethanol, and a mixed solvent of water and ethanol. These solvents may be used alone or in combination of two or more. In addition, when extracting with these organic solvents, in order to increase extraction efficiency, additives such as surfactants, enzyme preparations (pectinase, cellulase, protease, etc.) can be used as extraction solvents as long as the effects of the present invention are not impaired. You may add to

When using the mixed solvent of water and alcohol as an extraction solvent, it is preferable to make alcohol concentration into 10 to 90%. Specifically, a solvent such as water-containing ethanol having an ethanol concentration of 20 to 70%, and water-containing 1,3-butylene glycol having a concentration of 20 to 70% of 1,3-butylene glycol is suitably used, and the ethanol concentration is 30 to 30 The use of 60% hydrous ethanol is particularly preferred.

The amount of solvent used for extraction is usually 2 to 200 parts by weight, preferably 10 to 100 parts by weight, per 1 part by weight of the extraction raw material. If the amount of extraction solvent falls below this range, the extraction solvent may not spread over the entire extraction raw material, and the extraction efficiency may decrease, and if the amount of extraction solvent exceeds this range, the burden on removing the extraction solvent later is To increase.

The extraction temperature is a temperature equal to or lower than the boiling point of the solvent used for extraction, and is not particularly limited. For example, it may be heat extraction or normal temperature extraction.

The extraction time is 1 to 2 weeks in the case of cold extraction, and 30 minutes to 24 hours, preferably 1 to 10 hours in the case of heat extraction, but can be appropriately adjusted according to conditions such as the type of extraction solvent and extraction temperature. The number of extraction operations is not particularly limited, and may be one, or after the first extraction, fresh extraction solvent may be added again to perform the second and subsequent extraction operations. Moreover, you may perform extraction operation in multiple times using the same extraction solvent.

The extraction method may be any method commonly used in the art, and is performed using any apparatus at room temperature or under heating. Specifically, the extraction raw material is charged into an extraction treatment tank filled with an extraction solvent, and the soluble components are eluted while stirring occasionally as necessary, and then the residue is removed by filtration to obtain an extract.

The above-mentioned extract may be used as it is in the extracted solution, and if necessary, it may be further subjected to processing such as concentration, dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc. You may use it. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, lyophilization and the like, and used as a dried product.

The fat combustion promoting agent and hypothermia improving agent according to the present invention may be added with lactose, starch, cellulose, maltitol, dextrin, etc. in addition to crude drugs, vitamins, minerals, amino acids etc. insofar as the effects of the invention are not impaired. Additive, surfactant such as glycerin fatty acid ester, sucrose fatty acid ester, coating agent such as gelatin, pullulan, shellac, zein, oil such as wheat germ oil, rice germ oil, safflower oil, beeswax, rice bran wax, Waxes such as carnauba wax, sucrose, glucose, fructose, sweeteners such as stevia, saccharin and sucralose, and acidulants such as citric acid, malic acid and gluconic acid can be suitably contained. Examples of the herbal medicine include ginseng, American ginseng, rice field ginseng, propolis, agaricus, blueberries, eye blight, ginkgo biloba and extracts thereof and the like. Examples of vitamins include oil-soluble vitamins such as vitamin A, D, E and K, and water-soluble vitamins such as vitamins B1, B2, B6, B12, C, niacin, pantothenic acid, folic acid and biotin. Moreover, L-carnitine, L-citrulline, L-ornithine etc. are mentioned by an amino acid.

The intake of the passionflower extract according to the present invention can be appropriately determined according to the form, symptoms, age, body weight and the like. For example, when orally administered to adults, the daily dose is 1 to 1000 mg, preferably 5 to 500 mg, particularly preferably 10 to 100 mg.

The fat combustion promoting agent and hypothermia improving agent according to the present invention can be used in any of pharmaceuticals, quasi drugs, and foods. The method of administration includes oral administration (internal use) and transdermal administration (external use) and the like, so various dosage forms can be selected, and in particular, internal use is preferable. For example, common pharmaceutical forms such as powders, granules, tablets, coated tablets, capsules, syrups, pills, suspensions, solutions, emulsions, etc., beverages, gums, chocolate, candy, noodles, bread, cakes, Common food forms such as biscuits, mutton, jellies, canned, retort foods, meat and meat foods, seafood foods, margarines, butter, mayonnaise and the like can be adopted. Among them, beverages, capsules, tablets, granules and the like which can be easily adjusted in intake amount are preferable. The external preparations include dosage forms such as lotions, creams, emulsions, bath preparations, injections and suppositories.

According to the present invention, a fat burn promoting agent that exerts a fat burn promoting effect and a hypothermia improving effect by promoting differentiation into brown fat cells, increasing weight of brown fat tissue, enhancing UCP1 expression in brown fat cells and white fat cells A hypothermia improving agent can be provided.

Although an example is given in order to explain the present invention in detail, the present invention is not limited to this. The% of the content shown in the examples indicates% by weight.

(Preparation Example 1) Passion Flower 30% Ethanol Extract Passion Flower 20 g of whole grass dried (from France) is added 560 g of purified water and 240 g of ethanol, extracted for 1 week at room temperature, filtered and the filtrate The solution was concentrated and lyophilized to give 10.6 g of passionflower 30% ethanol extract.

Production Example 2 Passion Flower 60% Ethanol Extract Passion Flower 20 g of whole grass dried product (from France), add 320 g of purified water and 480 g of ethanol, extract for 1 week at room temperature, filter, and filter the filtrate Concentrated and lyophilized to give 10.6 g of passionflower 60% ethanol extract.

Production Example 3 Passionflower Hot Water Extract To 800 g of a mixture of dried passionflower stem and leaf (from France) was added 800 g of purified water, followed by extraction at 95 to 100 ° C. for 2 hours. The obtained extract was concentrated and dried to obtain 11.4 g of passionflower hot water extract.

Production Example 4 Passionflower Ethanol Extract To 800g of dried fruit (from France) of passionflower fruit, 800 g of ethanol is added and extracted for 1 week at room temperature, followed by filtration, and the filtrate is concentrated and lyophilized. 2.4 g of passionflower ethanol extract was obtained.

Beverage 1
<Prescription> Content (%)
1. Passion Flower 30% Ethanol Extract (Production Example 1) 0.05
2. Eye bright 20% ethanol extract 0.05
3. L-carnitine 1.0
4. Maltitol 8.0
5. Citric acid 0.35
6. Flavoring agent 0.1
7. Make the total amount 100 with purified water
<Manufacturing method>
Ingredients 1 to 6 are added to ingredient 7, stirred to dissolve, filtered, heat-sterilized and filled in a 100 mL aluminum can.
<Usage>
Take one bottle of this beverage daily.

Beverage 2
<Prescription> Content (%)
1. Passion Flower 60% Ethanol Extract (Production Example 2) 0.02
2. L-carnitine 0.3
3. Erythritol 8.0
4. Citric acid 0.3
5. Flavoring agent 0.1
6. Make the total amount 100 with purified water
<Manufacturing method>
Ingredients 1 to 5 are added to ingredient 6, stirred, dissolved, filtered, heat-sterilized and filled in a 100 mL aluminum can.
<Usage>
Take one bottle of this beverage daily.

Soft Capsule <Formulation> Content (%)
1. Passion Flower Hot Water Extract (Production Example 3) 1.0
2. Sunflower oil 90.0
3. Bee wax 6.0
4. Vitamin E 3.0
<Manufacturing method>
Ingredients 1 to 4 are mixed, 250 mg is filled in a coating composed of gelatin and glycerin, and after drying, a soft capsule is obtained.
<Usage>
Ingest 4 tablets per day.

Hard capsule <prescription> Content (%)
1. Passion Flower Ethanol Extract (Production Example 4) 5.0
2. Potato starch 92.0
3. Sucrose fatty acid ester 3.0
<Manufacturing method>
Ingredients 1 to 3 are mixed, and 250 mg is filled in a No. 2 hard capsule to obtain a hard capsule.
<Usage>
Ingest 4 tablets per day.

Tablet <prescription> Content (%)
1. Passion Flower 30% Ethanol Extract (Example 1) 10.0
2. Lactose 60.0
3. Reduced maltose starch syrup 20.0
4. Crystalline cellulose 5.0
5. Glycerin fatty acid ester 5.0
<Manufacturing method>
Ingredients 1 to 5 are mixed and pressed to obtain 0.3 g tablets.
<Usage>
Ingest 3 tablets per day.

Comparative Example 1 In the beverage of the conventional beverage Example 1, the passionflower 30% ethanol extract was replaced with water to obtain a conventional beverage.

(Experimental example 1) Examination of differentiation promoting effect to brown adipocytes Human subcutaneous adipose tissue-derived stem cells (ZENBIO) were seeded in 24 well plates at 5 × 10 ^{5 and} cultured in cell growth medium for 6 days. Gene expression analysis was carried out by culturing for 7 days after replacing the passion flower extract (Production Examples 1 to 4) with a white adipocyte differentiation induction medium dissolved to 0.25 mg / mL. The gene expression analysis evaluated the gene expression fluctuation | variation of UCP1, PGC1 alpha, CIDEA, and PDK4 which are genes expressed in brown fat cells by the real-time PCR method. The ratio of gene expression was calculated using the control group as 1.

Cell growth medium: 25 mM HEPES (Dojin Chemical), 3% GLUTAMAX-1 (GIBCO), 1% Insulin, Transferrin, Selenium, Ethanolamine Solution (GIBCO), 1% fetal bovine serum (SIGMA, 30 minutes at 56 ° C.) , 10 ng / mL FGF-basic (PEPROTECH), 0.4 μg / mL hydrocortison (WAKO), 50% αMEM (SIGMA) added to become 1% Antibiotic, Antimycotic (GIBCO), 50% DMEM (SIGMA) medium was used.

White adipocyte differentiation induction medium is 1 μM dexamethason (SIGMA), 0.5 mM 3-isobutyl-1-methylxanthine (SIGMA), 0.2 mM indomethacin (SIGMA), 10% fetal bovine serum (SIGMA), 10 μg / mL insulin ( SIGMA), 33 μM biotin (SIGMA), DMEM (SIGMA) medium supplemented to 1% Antibiotic, Antimycotic (GIBCO) was used.

Table 1 shows the results of the expression ratio of brown fat cell expressed genes. Addition of passionflower extract enhanced the gene expression of brown adipocyte-expressing genes UCP1, PGC1α, CIDEA, and PDK4. That is, it has been shown that the passionflower extract has an effect of promoting differentiation into brown fat cells.

(Experimental example 2) Examination of brown fat tissue increasing action of obese model mice After preparing 8-week-old female C57BL / 6J mice for preliminary breeding, obese-extracted obese model mice are prepared and a non-added group and passion flower The extract (Production Examples 1 to 4) was divided into groups. In addition, mice subjected to a sham operation were provided as a control group. After the treatment, the experimental diet shown in Table 2 was given, and after rearing for 8 weeks, brown adipose tissue was excised and weighed. The brown adipose tissue weight per body weight of each individual was calculated and evaluated.

The results of brown adipose tissue weight are shown in Table 3. Feeding passionflower extract increased brown adipose tissue weight. That is, it has been shown that the passionflower extract has an effect of increasing brown adipose tissue.

(Experimental Example 3) Examination of UCP1 Increasing Effect on Brown Adipocytes of Obese Model Mice Gene expression analysis and immunostaining were performed using brown adipose tissue collected in the same manner as in Experimental Example 2. Gene expression analysis evaluated gene expression fluctuation of UCP1, PGC1α, CIDEA, PDK4 expressed in brown fat cells by real-time PCR. The ratio of gene expression was calculated using the control group as 1. The amount of protein was evaluated by staining with red fluorescence using Anti-UCP1 (CALBIOCHEM) for immunostaining.

The results of the expression ratio of brown fat cell expressed genes are shown in Table 4. Feeding passionflower extract to obese model mice enhanced the gene expression of UCP1, PGC1α, CIDEA and PDK4 in brown adipose tissue. In immunostaining, since the fluorescence intensity of the red stained UCP1 was increased by giving the passionflower extract, it can be said that UCP1 protein of brown fat cells increased. That is, it was shown that the passion flower extract has an effect of increasing UCP1 of brown fat cells.

(Experimental Example 4) Examination of UCP1 Increasing Action in White Adipocytes Human subcutaneous adipose tissue-derived stem cells were differentiated into white adipocytes in the same manner as in Experimental Example 1. Then, cell maintenance medium (passion flour extract was dissolved to 0.75 mg / mL (10% fetal bovine serum (SIGMA), 10 μg / mL insulin (SIGMA), 33 μM biotin (SIGMA), 1% Antibiotic, After replacing with DMEM (SIGMA) medium added to become Antimycotic (GIBCO), the cells were cultured for 4 days, and gene expression analysis and immunostaining were performed. Gene expression analysis evaluated gene expression fluctuation of UCP1, PGC1α, CIDEA, PDK4 expressed in brown fat cells by real-time PCR. The ratio of gene expression was calculated using the control group as 1. The amount of protein was evaluated by staining with red fluorescence using Anti-UCP1 (CALBIOCHEM) for immunostaining.

The results of the expression ratio of brown fat cell expressed genes are shown in Table 5. The addition of passionflower extract enhanced the gene expression of UCP1, PGC1α, CIDEA and PDK4 in cells differentiated into white adipocytes. In immunostaining, it is possible to say that UCP1 protein was increased because addition of the passionflower extract increased the fluorescence intensity of red stained UCP1. That is, it was shown that the passionflower extract has an effect of increasing UCP1 of white fat cells.

(Experimental Example 5) Examination of UCP1 Increasing Effect on White Adipocytes of Obese Model Mice Inguinal white adipose tissue was collected from a mouse bred by the same method as in Experimental Example 2, and gene expression analysis and immunostaining were performed. Gene expression analysis evaluated the gene expression change of UCP1 by the real-time PCR method. The ratio of gene expression was calculated using the control group as 1. The amount of protein was evaluated by staining with red fluorescence using Anti-UCP1 (CALBIOCHEM) for immunostaining.

The results of the expression ratio of brown fat cell expressed genes are shown in Table 6. Feeding passionflower extract to obese model mice enhanced UCP1 gene expression in white adipose tissue. In immunostaining, since the fluorescence intensity of the red stained UCP1 was increased by providing the passionflower extract, it can be said that the UCP1 protein of white fat cells was increased. That is, it was shown that the passionflower extract has an effect of increasing UCP1 of white fat cells.

(Test Example 6) Examination of hypothermia improvement action Test group 1 so that the average value of the oral temperature becomes equal for every 10 men and women (oral temperature 35.9 to 36.1 ° C.) with lower deep body temperature, which is lower. The test group 2 is divided into a test group 1 using a beverage containing 30% ethanol extract of passionflower (Example 1), and a test group 2 using a conventional beverage (Comparative Example 1), 100 mL once a day Was ingested for 1 month. The oral temperature was measured with a thermometer before and after the test.

The oral temperatures before and after the test are shown in Table 7. Although there was no change before and after drinking in Comparative Example 1, the oral temperature increased before and after drinking in Example 1. That is, it has been shown that the passionflower extract has an effect of improving hypothermia by raising core body temperature. During the trial, there was no subject who got ill, and there was no problem in safety. In addition, there was no problem with the deterioration of prescription ingredients.

Also, instead of the beverage (Example 1), a beverage (Example 2), 4 soft capsules (Example 3) per day, 4 hard capsules (Example 4) per day, tablets (implementation The same effect was observed when 3 tablets of Example 5) were ingested daily.

The present invention relates to a fat combustion promoter and a hypothermia improving agent characterized by containing passionflower extract. Therefore, it is useful for medicines, quasi-drugs, foods, etc. for the purpose of improvement and prevention of obesity, metabolic syndrome and hypothermia.

Claims (1)

The hypothermia improving agent which contains the hydrous ethanol extract whose ethanol concentration is 30 to 60% of passionflower whole grass as an uncoupling protein 1 (UCP1) increase agent in white fat cells .