JP2013032298A - Aqueous solvent extract of marine humus and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new anti-obesity and/or anti-arteriosclerotic composition.SOLUTION: The anti-obesity and/or anti-arteriosclerotic composition contains an active constituent which is an aqueous solvent extract of marine humus.

Description

The present invention relates to a novel use of an aqueous solvent extract of marine humus.
Specifically, the present invention relates to an anti-obesity and / or anti-arteriosclerotic composition comprising an aqueous solvent extract of marine humus as an active ingredient.

  Marine humus was built in the ancient years of 5 million to tens of millions of years, where seaweeds, algae, plants, seafood and other organisms were buried deep in the seabed and accumulated over time. It is thought that the stratum was raised by crustal deformation.

  In the marine humus soil, the above-mentioned seaweed, algae, plants, seafood and other organisms as marine humic substances are decomposed by microorganisms in the soil; fermented products fermented in the soil; It is known to contain various organic substances and minerals such as digested products digested by soil organisms; and the above decomposed products, fermented products, and synthesized products synthesized from microorganisms with microorganisms.

Moreover, most of the organic substances contained in the marine humus are the marine humic substances described above.
When the marine humus is treated with an alkali such as sodium hydroxide or sodium pyrophosphate, a deep red-brown suspension containing 60-80% of the marine humic substances contained in the marine humus is obtained.

The insoluble component obtained by separating this suspension into an aqueous solution and an insoluble component by a conventional method is called humin. This humin is a mixture of high-molecular organic substances that are insoluble not only in alkalis but also in acids, and has a property of strongly binding to inorganic components of soil.
Further, when an acid having a pH of about 1 to 2 is added to the solution obtained by adding alkali to make it acidic, a blackish brown cotton-like precipitate is formed. This insoluble substance is called humic acid.
Humic acid is a substance having a molecular weight of up to about 300,000, and the properties as humic substances are attributed to this humic acid.

  The yellow supernatant obtained by suspending marine humus in water is called fulvic acid, which is soluble in alkali and acid. This fulvic acid is a mixture of substances having a relatively small molecular weight of about 2,000 to 50,000. This fraction is a fraction in fractionation operation, and does not represent an essential difference in the mixture of organic substances contained in the fraction. Although this fulvic acid has been studied for a long time, its essence is not well understood (Non-patent Documents 1 and 2).

  Examples of the fulvic acid include stepped fulvic acid provided by the Japanese Humic Society and Chikugo River fulvic acid provided by the Saga University Research Center.

  So far, regarding the physiological activity of marine humic substances, antibacterial / bactericidal action, antitumor effect and the like have been reported, but no other physiological action has been reported (Non-Patent Documents 3 to 5).

  In recent years, lifestyle-related diseases such as arteriosclerosis are increasing rapidly. The rapid increase in lifestyle-related diseases is thought to be caused by obesity accompanying the recent westernization of dietary habits. In obese humans, adipocytes or adipose tissue tend to enlarge.

  Adipocytes and adipose tissue not only accumulate fat but also secrete physiologically active substances (adipocytokines). Examples of adipocytokines that are secreted proteins derived from adipose tissue include leptin, adiponectin, and plasminogen activator inhibitor-1 (PAI-1).

  For example, it is known that arteriosclerosis is promoted when the amount of adiponectin secretion decreases. Moreover, when the secretion amount of PAI-1 increases, the fibrinolytic activity is inhibited, and a tendency to form a thrombus (prothrombotic) occurs. As a result, it promotes the development of arteriosclerosis and subsequent thromboembolic diseases. In general, it is known that when adipocytes are enlarged, that is, obese, the amount of adiponectin secreted decreases. Thus, obesity is believed to be closely related to the development of arteriosclerosis and secondary thromboembolic diseases.

Regarding the regulation of adipocytokine expression, Nagasawa A. et al. Reported that the expression of PAI-1 gene in testicular epithelial white adipose tissue was suppressed by protein extracted from soybean rather than animal protein (casein), and the expression of adiponectin gene and blood It has been reported that the concentration of medium adiponectin is increased (Non-patent Document 6).
There are no reports on the regulation of adipocytokine expression by other food components.
On the other hand, it is known that certain mineral components inhibit PAI-1 secretion from cultured vascular endothelial cells.

  The progression of arteriosclerosis involves the migration and proliferation of vascular smooth muscle cells into the intima, and the reception of fibrinolytic factors in the proliferation of vascular smooth muscle cells in the presence of tumor cells (mouse melanoma cells) The body is important (Non-patent Document 7).

The migration ability of vascular smooth muscle cells is changed by PAI-1 and adiponectin secreted from adipocytes.
PAI-1 promotes the migration ability of vascular smooth muscle cells by binding to vitronectin, an extracellular matrix, and adiponectin suppresses the migration ability of vascular smooth muscle cells through signal transduction in vascular smooth muscle cells It has been known.

Teruo Matsunaka: Basics of Soil Science-Generation, Function, Fertile Soil, Environment-, Agriculture, Mountain and Fishing Village Cultural Association, 47-49 (2003) Kuma Kuma, Sadao Shoko et al .: New Soil Science, Asakura Shoten, 40-49 (1989) Kodama H., Denso and Nakagawa T .: Protection against atypical Aeromonas salmonicida infection in carp (Cyprinus carpio L.) by oral administration of humus extract, J. Vet. Med. Sci., 69 (4), 405-408 (2007 ) Kodama H. and DENSO: Antitumor Effect of Humus Extract on Murine Transplantable L1210 Leukemia, J. Vet. Med. Sci., 69 (10), 1069-1071 (2007) Kodama H., Denso, Okazaki F. and Ishida S .: Protective effect of humus extract against Trypanosoma brucei infection in mice., J. Vet. Med. Sci., 70 (11): 1185-90 (2008) Nagasawa A., Fukui K., Kojima M., Kishida K., Maeda N., Nagaretani H., Hibuse T., Nishizawa H., Kihara S., Waki M., Takamatsu K., Funahashi T. and Matsuzawa Y .: Divergent effects of soy protein diet on the expression of adipocytokines., Biochem Biophys Res Commun. 311 (4): 909-14 (2003) Ueshima S., Fukao H., Okada K. and Matsuo O .: Growth inhibition of vascular smooth muscle cells derived from urokinase receptor (u-PAR) -deficient mice in the presence of carcinoma cells., Thromb Res. 113 (1) : 41-9 (2004)

  The problem to be solved by the present invention is to provide a novel anti-obesity and / or anti-arteriosclerotic composition.

  The present inventors have investigated the effect of an aqueous solvent extract of marine humus soil on adipocytes, and the effect on proliferation and migration ability of vascular smooth muscle cells in the presence of adipocytes. As a result of intensive research efforts aimed at clarifying the results, it was surprisingly found that an aqueous solvent extract of marine humus soil has anti-obesity and anti-arteriosclerotic effects, leading to the completion of the present invention. It was.

  According to the present invention, there is provided an anti-obesity and / or anti-arteriosclerotic composition, wherein the active ingredient is an aqueous solvent extract of marine humus.

  Moreover, according to this invention, the said aqueous solvent is the said composition which is water or a 50 volume% or less lower alcohol aqueous solution.

  Furthermore, according to the present invention, a food or health food comprising the composition according to the present invention is provided.

  According to the present invention, an aqueous solvent extract of marine humus can be safely used as an anti-obesity and / or anti-arteriosclerotic composition.

It is a graph which shows the influence on the oil droplet accumulation amount by addition of the aqueous solvent extract of marine humus soil. It is a schematic diagram showing a method for examining the migration ability of vascular smooth muscle cells in the presence of adipocytes. It is a graph which shows the change of the migration ability of the vascular smooth muscle cell by the differentiation stage of the coexisting fat cell. It is a graph which shows the influence which the aqueous-solvent extract of marine humus soil has on the migration ability of the vascular smooth muscle cell in presence of an adipocyte. It is a graph which shows the influence which the aqueous solvent extract of marine humus soil has on the active type PAI-1 secreted from an adipocyte. It is a graph which shows the influence which the aqueous solvent extract of marine humus soil has on the adiponectin secreted from an adipocyte. It is a graph which shows the influence which the aqueous solvent extract of marine humus soil has on the activated PAI-1 secreted from the fat cell in the presence of vascular smooth muscle cells. It is a graph which shows the influence which the aqueous solvent extract of marine humus soil has on the adiponectin secreted from the fat cell in the presence of vascular smooth muscle cells. It is a graph which shows the influence which the aqueous solvent extract of marine humus soil has on the t-PA activity in an adipocyte culture solution. It is a graph which shows the influence which the aqueous solvent extract of marine humus soil has on u-PA activity in an adipocyte culture solution.

In the present invention, an aqueous solvent extract of marine humus is used.
The marine humus soil used in the present invention is not particularly limited, but the marine humus soil layer present at a depth of 0.5 to 250 m from the seabed, or the rippling surface of the marine humus soil layer raised by crustal deformation. Or the marine humus soil extract | collected from the marine humus soil layer which exists in the depth of 5-30 m of a wetland can be used.

  Examples of such marine humus include, for example, powdered marine humus from Ariake Sea (Lot. No. 08-02) manufactured by Marinex.

Examples of the aqueous solvent include water; an acidic aqueous solution such as dilute hydrochloric acid; and an aqueous solution of 50% by volume or less of a lower alcohol such as aqueous ethanol.
Water from which chlorine is removed is preferable. The method for removing chlorine is not particularly limited, and examples thereof include a method of leaving at room temperature for several days, a method using a reducing agent such as ascorbic acid, a method using an adsorbent such as activated carbon or an ion exchange resin, and the like. Further, ion exchange distilled water or Millipore water is more preferable.

The term “lower alcohol” used in the present invention means methanol, ethanol, propanol, butanol or isomers of these alcohols or a mixture of these alcohols.
The pH of the aqueous solvent is not particularly limited, but is preferably in the range of pH 6.0 to 8.0, more preferably in the range of pH 6.3 to 7.5.

The method for obtaining the aqueous solvent extract of marine humus used in the present invention is not particularly limited, but a method including the following steps (1) to (3) is preferred.
(1) Repeated steps of stirring and leaving a mixture of marine humus soil and aqueous solvent (1: 5 to 1:10 (weight ratio));
(2) solids removal step;
(3) A step of obtaining a supernatant by allowing the obtained suspension containing fine insoluble matter to stand at ambient temperature for a long period of time.

In the step (1), first, in a container such as a water tank, 5 to 10 times (weight ratio) of an aqueous solvent is added to the marine humus soil while stirring, and suspended. Get things.
The resulting suspension is then allowed to stand at room temperature.
As used herein, “room temperature” means a room temperature of 15 to 45 ° C., preferably 20 to 40 ° C., more preferably 25 to 35 ° C.
The aqueous solvent extract of the marine humus soil of the present invention is prepared by repeating the above stirring and standing.
Stirring may be performed to such an extent that the marine humus settled at the bottom of the container is diffused into the aqueous solvent, and may be either continuous or intermittent. This process can last for a week or more.

  The method for separating the solid matter in the above step (2) is not particularly limited as long as it is a method capable of separating the marine humus soil and the liquid, or the precipitate is separated by decantation after standing, Alternatively, it can be separated by centrifugation or the like.

  In the method of separating the precipitate by decanting, the suspension is allowed to stand at 25 to 35 ° C. for about 150 to 240 hours to precipitate marine humus, and then only the supernatant is collected by decanting. In order to remove the solid matter more reliably, the suspended matter may be filtered using a cloth or the like. The supernatant thus obtained has a pH of about 2.5 to 3.0.

  Step (3) is performed by leaving the obtained supernatant at ambient temperature for a long period of time. The long term may be about 180 to 500 days, preferably about 300 to 400 days. During this standing, the temperature may be constant or may vary. Usually, if it hold | maintains for 30 to 60 days at the temperature of 25-35 degreeC, and hold | maintains for 30 to 60 days at the temperature of 5-15 degreeC, the aqueous-solvent extract of marine humus soil more preferable to use by this invention can be obtained. it can.

The liquid obtained by the above standing may be further filtered. The filtration may be performed to such an extent that the suspended matter in the liquid can be removed. For example, the filtration can be performed using two types of membrane filters having a pore diameter of 40 to 100 μm and 10 to 40 μm.
The aqueous solvent extract of marine humus soil obtained by the above method is a yellow to light brown substantially odorless liquid having a pH of 2.0 to 3.5 and an electric conductivity of 350 to 500 mV.

Further, the aqueous solvent extraction of the above-mentioned marine humus can be carried out actively in a short time using a ultrasonic homogenizer or the like.
That is, 3 to 12 parts by weight of distilled water, preferably 4 to 8 parts by weight, and more preferably 6 parts by weight of the aqueous solvent extract of the above-mentioned marine humus soil with respect to 1 part by weight of the marine humus soil. And sonicated with an ultrasonic homogenizer Ira VCX-130 (SONICS & MATERIALS Inc ,; 130 Watt, 20 kHz, 30% amplitude) for 10 minutes. This sonication solution is centrifuged at 1000 rpm for 5 minutes, and the resulting supernatant is filtered using Stella Disk (0.2 μm), and the obtained filtrate (pH 2.83) is removed from marine humus soil. It can also be used as an aqueous solvent extract.

  In addition, this aqueous solvent extract of marine humus soil has a weight after freeze-drying of 0.5 to 1 g per 100 ml of the marine humus soil aqueous solvent extract, and is adsorbed through the DAX-8 resin column. The weight after freeze-drying of (fulvic acid) is 0.001 g or less per 100 ml of the aqueous solvent extract of marine humus soil.

Further, the aqueous solvent extract of the marine humus can be further subjected to a purification treatment.
For the purification treatment, a chromatographic method, an ion exchange chromatographic method and the like known to those skilled in the art can be employed alone or in combination in addition to the above-described partition extraction with a solvent. For example, as a chromatographic method, a column chromatography, a thin layer chromatography, a high performance liquid chromatography, a centrifugal liquid chromatography or the like using a normal phase or reverse phase carrier or an ion exchange resin, or a combination thereof is performed. Is mentioned. In this case, purification conditions such as a carrier and an elution solvent can be appropriately selected according to various chromatographies.

  Moreover, the aqueous solvent extract of the marine humus can be further subjected to a concentration treatment. Concentration is preferably performed under reduced pressure. Concentration may be performed until the extract is dry.

  Therefore, the aqueous solvent extract of marine humus soil in the present invention is an aqueous solvent extract of marine humus soil, its concentrate, its concentrated dried product, lyophilized product, the above-mentioned terraced fulvic acid and Chikugo River Although any fulvic acid including fulvic acid is meant, the extract according to the present invention can be used as it is without purification, and constitutes a part of the present invention.

  The aqueous solvent extract of the marine humus soil according to the present invention is an extract obtained by extracting the marine humus soil with water according to the above method, and can be used as a soft drink as it is. The product can be used as a food additive or composition.

  The extract of the present invention may be used as it is, diluted with an appropriate medium, or by a method known in the field of pharmaceutical production, such as a solid agent (for example, powder, granule, tablet, capsule), semi-solid agent or It can be formulated into various pharmaceutical forms such as liquids.

  In these pharmaceutical forms, an appropriate medium may be added. Such media include pharmaceutically acceptable excipients such as binders or disintegrants.

  Examples of excipients include sugar alcohols such as erythritol, maltitol, mannitol, sorbitol, xylitol, and lactitol, purified sucrose, sucrose, trehalose, lactose, reduced maltose water candy, powdered reduced maltose water candy, glucose, maltose, and the like. Examples include saccharides, corn starch, crystalline cellulose, powdered cellulose, calcium monohydrogen phosphate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, calcium lactate, precipitated calcium carbonate, light anhydrous silicic acid, hydrous silicon dioxide, silicon dioxide and the like.

  Examples of the binder include gum arabic powder, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), cellulose derivatives such as methylcellulose, carboxymethylcellulose, carboxymethylethylcellulose, povidone (PVP), vinylpyrrolidone copolymer Examples include coalescence (copolyvidone), polyvinyl alcohol (PVA), acrylic polymer, pullulan, dextrin, pregelatinized starch, hydroxypropyl starch, tragacanth powder, crystalline cellulose, low-substituted hydroxypropylcellulose (L-HPC), and the like. .

  Examples of the disintegrant include croscarmellose sodium, low-substituted hydroxypropyl cellulose, carmellose calcium, crospovidone, corn starch, carboxymethyl starch sodium, hydroxypropyl starch, and partially pregelatinized starch.

  In the preparation, further additives such as a lubricant, a fluidizing agent, a coloring agent, a pH adjusting agent, a sweetening agent, a fragrance, and an antiseptic may be used. Other carrier components or additives include lubricants (eg, magnesium stearate, calcium stearate, talc, sucrose fatty acid ester, glycerin fatty acid ester, hydrogenated oil, macrogol 6000, etc.), fluidizing agents (eg, light Silicic anhydride, hydrous silicon dioxide, kaolin, etc.), antioxidants (eg, dibutylhydroxytoluene (BHT), propyl gallate, butylhydroxyanisole (BHA), tocopherol, citric acid, etc.), preservatives (paraoxybenzoic acid ester) Etc.),

Colorant (for example, riboflavin, vitamin B12, titanium oxide, yellow ferric oxide, ferric oxide, edible red No. 2, edible red No. 3, edible red No. 102, edible red No. 104, edible red No. 105, edible red 106 No., Edible Yellow No. 4, Edible Yellow No. 5, Edible Green No. 3, Edible Blue No. 1, Edible Blue No. 2, Copper Chlorophyll Sodium, Copper Chlorophyll, etc.), pH regulators (eg, sodium hydroxide, sodium citrate, Hydrochloric acid, sodium bicarbonate, sodium carbonate, calcium lactate, phosphoric acid, dipotassium phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate)

Sweetening agents (eg, sucrose, mannitol, D-sorbitol, xylitol, aspartame, stevia, dipotassium glycyrrhizinate, acesulfame K, sucralose, etc.), flavors (eg, L-menthol, peppermint oil, eucalyptus oil, orange oil, Clove oil, turpentine oil, fennel oil, vanillin, etc.), surfactant (polyoxyethylene hydrogenated castor oil, glyceryl monostearate, sorbitan fatty acid ester (such as sorbitan monostearate, sorbitan monolaurate), polyoxyethylene polyoxypropylene, Polysorbates, sodium lauryl sulfate, macrogol, sucrose fatty acid esters, etc.), plasticizers (triethyl citrate, polyethylene glycol, triacetin, cetanol, etc.), flavoring agents or flavorings (Such as menthol), adsorbents, preservatives, wetting agents, and antistatic agents.

  If necessary, the solid agent is a water-soluble base (or polymer) such as HPC, HPMC, or povidone, an insoluble base (or polymer) such as ethyl cellulose, an enteric base, a gastric base, or a saccharide. It may be coated.

  The solid agent can be produced by a conventional method. The active ingredient and the carrier component may be mixed to prepare a powder. Usually, the active ingredient and the carrier component are granulated, and if necessary, the granulated product is sized and granulated (fine granule or granule). A bare tablet can be prepared by tableting a mixture containing the granulated product (especially a mixture of the granulated product and the carrier component). Capsules can be prepared by filling capsules with the granules.

  Granulation can be performed by a conventional method, for example, stirring granulation method, fluidized bed granulation method, extrusion granulation method, dry granulation method and the like. A preferred granulation method is a fluidized bed granulation method. In granulation, an active ingredient and a carrier component are often granulated using a solution containing a binder. For example, by spraying a solution containing a binder on a fluidized bed of the active ingredient and the carrier component. Can be granulated. The coating preparation can be obtained by spraying a coating agent containing a coating base onto an uncoated preparation (elementary granule, plain tablet, etc.) using a film coating machine.

  In the liquid agent, an aqueous medium (purified water, ethanol-containing purified water, etc.), alcohols (ethanol, glycerin, etc.), water-soluble polymers, etc. can be used as the carrier component. As the carrier component, purified water, ethanol-containing purified water and the like are often used. As the carrier component of the semi-solid preparation, an oily base (lipid such as vegetable oil, petrolatum, liquid paraffin, etc.), a hydrophilic base (emulsion base) and the like can be used. Examples of additives include disintegration aids, antioxidants or antioxidants, surfactants, emulsifiers, dispersants, suspension agents, solubilizers, thickeners, pH adjusters or buffers, preservatives or Preservatives (such as parabens), bactericides or antibacterial agents, antistatic agents, corrigents or masking agents (such as sweeteners), cooling agents, coloring agents, flavoring agents, or fragrances.

  The liquid preparation can be prepared by dissolving or dispersing each component in a carrier component, filtering or sterilizing if necessary, and filling a predetermined container. The semi-solid preparation can also be prepared by a conventional method, for example, by mixing each component and a carrier component, sterilizing if necessary, filling a predetermined container, or applying to a substrate.

The amount of the aqueous solvent extract to be used varies depending on its form, but 100 ml to 500 ml at a time is appropriate for a liquid, and 0.1 g to 5 g is appropriate for a solid.
The water extract of marine humus soil is also approved as a soft drink and can be used safely because it is extremely low in toxicity or negligible.

When the extract according to the present invention is used as a solid agent or a semisolid agent, the amount used varies depending on age, symptoms, etc., but it is 0.1 to 1 g per day for adults 1 to 3 times a day. Can be used to some extent.
Moreover, when using as a liquid agent, about 10 ml-500 ml per time can be used for adults 1 to 3 times a day.

  Moreover, what added the composition by this invention to a foodstuff as an active ingredient can be utilized as a foodstuff or health food.

Health food means food with a more positive meaning than normal food for the purpose of health, health maintenance, promotion, etc. Examples include teas, nutritional drinks, and soups.
In the production process of these foods or to the final product, the above-mentioned extract can be added to make a health food.

  The food or health food is used in an amount that does not adversely affect the taste and appearance, for example, in the range of about 0.1 g to 10 g of the above dried solids per 1 kg of the target food or health food, The water-soluble solvent extract according to the present invention can be used in place of water used in the health food production process.

  Hereinafter, examples according to the present invention will be described more specifically with reference to the drawings. However, the present invention is not limited to the following examples. In the description of this specification and the description of the drawings, the descriptions “Control” and “Cont.” Mean “control”.

Material As the marine humus, a powdery marine humus (Lot. No. 08-02) from Ariake Sea manufactured by Marinex was used.

Production Example 1
Aqueous solvent extract of marine humus soil An aqueous solvent extract of marine humus soil was mixed with 3.0 g of powdered marine humus soil manufactured by Marinex Co., Ltd. using 18.0 g of distilled water. Sonication was performed with a sonic homogenizer Ira VCX-130 (SONICS & MATERIALS Inc .; 130 Watt, 20 kHz, 30% amplitude) for 10 minutes. This sonication solution was centrifuged at 1000 rpm for 5 minutes, and 11.0 g of the obtained supernatant was filtered using Stella Disk 25 (manufactured by Kurashiki Boseki Co., Ltd .; Lot. No. 210907). The product was used as a sample of marine humus soil aqueous solvent extract.

  3T3-L1 preadipocytes were obtained from the Human Science Research Foundation (JCRB9014, Osaka, Japan). Mouse-derived vascular smooth muscle cells are described in YongZhong Hou et al. (Hou Y., Okada K., Okamoto C., Ueshima S. and Matsuo O .: c-Myc is essential for urokinase plasminogen activator expression on hypoxia-induced vascular smooth muscle cells. Cardiovasc Res., 75 (1): 186-94 (2007)).

Example 1
Effect of marine humus soil extract (hereinafter also simply referred to as humus soil) on the amount of oil droplet accumulation The marine humus soil extract was added to the differentiation maintenance medium. The marine humus soil extract was added to the differentiation maintenance medium so that the medium volume was 1% by volume. As a control, sterilized distilled water was added to the differentiation maintenance medium so that the medium volume was 1% by volume. 3T3-L1 cells on differentiation day 2, 4, 6, and 8 were stained with Oil-Red O. After dye extraction, absorbance measurement at a wavelength of 492 nm (OD492 nm) was performed with a microplate reader to evaluate the amount of accumulated oil droplets. The results are shown in FIG. Absorbance (OD 492 nm) is shown on the vertical axis. The absorbance values on the 8th day of differentiation were 0.860 ± 0.489 for the control and 0.626 ± 0.441 for the marine humus extract, and the addition of the marine humus extract significantly suppressed the accumulation of oil droplets compared to the control (Fig. 1).

Example 2
Examination of migration ability of vascular smooth muscle cells in the presence of adipocytes Attach an up chamber in which vascular smooth muscle cells are seeded to the down chamber where adipocytes from day 0 to day 8 of differentiation exist, and after 4 hours The number of cells migrating to the back side was counted (FIG. 2).
Adipocytes were prepared in the down chamber every 2 days of differentiation, and 0.3 × 10 ⁵ vascular smooth muscle cells were seeded in the up chamber. After seeding, cells that passed through the membrane in 4 hours were fixed and stained, and the number of cells that migrated to the back of the membrane was counted to evaluate the migration ability of vascular smooth muscle cells. The results are shown in FIG. The number of migrating cells per field is shown on the vertical axis. The number of migrating cells of vascular smooth muscle cells in the presence of adipocytes after the 4th day of differentiation was significantly higher than the number of migrating cells of vascular smooth muscle cells in the presence of adipocytes on the 0th day of differentiation (FIG. 3).

  The migration ability of vascular smooth muscle cells was evaluated in the presence of adipocytes cultured for 2 days in a differentiation maintenance medium containing marine humus soil extract in the down chamber. Furthermore, when culturing adipocytes in the down chamber, change to a differentiation maintenance medium containing marine humus soil extract every 2 days of differentiation, and evaluate the migration ability of vascular smooth muscle cells in the presence of adipocytes until the 8th day of differentiation. did. Sterile distilled water was used as a control. The results are shown in FIG. The number of migrating cells per field is shown on the vertical axis. By the marine humus soil extract, the migration ability of vascular smooth muscle cells in the presence of differentiated adipocytes was significantly lower than the control on the 4th and 8th days of differentiation (FIG. 4).

Example 3
Effect of marine humus soil extract on PAI-1 and adiponectin secretion from adipocytes The medium of 3T3-L1 adipocytes after differentiation induction was replaced with a differentiation maintenance medium containing 1% by volume of marine humus soil extract. A differentiation maintenance medium containing 1% by volume of sterile distilled water was used as a control. The day of replacement with this differentiation maintenance medium was defined as day 0 of differentiation, and the medium was replaced with the same medium every two days. Then, the culture medium on the 8th day of differentiation was collected, and active PAI-1 in the culture medium was quantified using an ELISA kit. The results are shown in FIG. The vertical axis represents the concentration of active PAI-1. The amount of active PAI-1 in the culture medium on the 8th day of differentiation did not change with respect to the control cultured with sterilized distilled water added to the differentiation maintenance medium (FIG. 5).

  The medium of 3T3-L1 adipocytes after differentiation induction was replaced with a differentiation maintenance medium containing 1% by volume of marine humus soil extract. A differentiation maintenance medium containing 1% by volume of sterile distilled water was used as a control. The day of replacement with this differentiation maintenance medium was defined as day 0 of differentiation, and the medium was replaced with the same medium every two days. Then, the culture medium on the 8th day of differentiation was collected, and adiponectin in the culture medium was quantified using an ELISA kit. The results are shown in FIG. The vertical axis represents the concentration of adiponectin. The amount of adiponectin in the culture medium on the 8th day of differentiation was significantly increased compared to the control cultured by adding sterile distilled water to the differentiation maintenance medium (FIG. 6).

  A marine humus soil extract was added to the adipocyte differentiation maintenance medium, and after adipocytes were cultured in the down chamber every 2 days for 8 days, an up chamber in which vascular smooth muscle cells were seeded was attached. The migration ability of vascular smooth muscle cells was evaluated, and the culture medium at that time was collected to quantify active PAI-1 in the medium. The amount of active PAI-1 in the medium when evaluating the migration ability of vascular smooth muscle cells using a differentiation maintenance medium containing 1% by volume sterile distilled water was used as a control (Cont.). The results are shown in FIG. The vertical axis represents the concentration of active PAI-1. The amount of active PAI-1 in the medium collected 4 hours after the installation of the Up chamber showed a tendency to decrease by the marine humus soil extract as compared with the control (FIG. 7).

  A marine humus soil extract was added to the adipocyte differentiation maintenance medium, and after adipocytes were cultured in the down chamber every 2 days for 8 days, an up chamber in which vascular smooth muscle cells were seeded was attached. The migration ability of vascular smooth muscle cells was evaluated, the culture medium at that time was collected, and adiponectin in the medium was quantified. Adiponectin cultured in a differentiation maintenance medium containing 1% by volume of sterile distilled water every two days for 8 days was present, and the amount of adiponectin in the medium when the vascular smooth muscle cell migration ability was evaluated was used as a control (Cont .). The results are shown in FIG. The vertical axis shows the concentration of adiponectin. The amount of adiponectin in the medium collected 4 hours after installation of the Up chamber was significantly reduced compared to the control (FIG. 8).

Example 4
Effect of marine humus soil extract on plasminogen activator (PA) activity in adipocyte culture medium Cultured with the addition of marine humus soil extract in adipocyte differentiation maintenance medium. The collected medium was analyzed by fibrin enzyme chromatography. Then, the t-PA activity in the adipocyte culture was compared with the t-PA activity (control) when sterilized distilled water was added to the differentiation maintenance medium. The results are shown in FIG. The t-PA relative activity (vertical axis) is the percentage of t-PA activity in the culture solution when the marine humus extract (humus soil) is added to the differentiation maintenance medium when the control is 100%. Show. The t-PA relative activity was significantly increased compared to the control (FIG. 9).

  The culture was performed by adding the marine humus soil extract to the differentiation maintenance medium for adipocytes, and the medium collected on the 8th day of differentiation was analyzed by fibrin enzyme chromatography. Then, the u-PA activity in the adipocyte culture was compared with the u-PA activity (control) when sterilized distilled water was added to the differentiation maintenance medium. The results are shown in FIG. The u-PA relative activity (vertical axis) is the percentage of u-PA activity in the culture solution when the marine humus extract (humus soil) is added to the differentiation maintenance medium when the control is 100%. Show. The u-PA relative activity was significantly increased compared to the control (FIG. 10).

  According to the present invention, an aqueous solvent extract of marine humus soil having no cytotoxicity can be safely used as an anti-obesity and / or anti-arteriosclerotic composition.

Claims (6)

  An anti-obesity composition, wherein the active ingredient is an aqueous solvent extract of marine humus.   An anti-arteriosclerotic composition, wherein the active ingredient is an aqueous solvent extract of marine humus.   The composition according to claim 1 or 2, wherein the marine humus is marine humus from Ariake Sea.   The composition according to claim 1 or 2, wherein the extract is stepped fulvic acid or Chikugogawa fulvic acid.   The composition according to any one of claims 1 to 4, wherein the aqueous solvent is water or a 50% by volume or less lower alcohol aqueous solution.   A food or health food comprising the composition according to any one of claims 1 to 5.