JP5566619B2 - Method for producing mushroom-derived active ingredients - Google Patents

Description

  The present invention relates to a method for producing an adipocyte differentiation promoting substance from mushrooms. In particular, the present invention provides an adipocyte differentiation promoting substance that activates PPARγ (peroxisome proliferator-activated receptor γ), which is a nuclear receptor, from mushrooms and promotes differentiation into adipocytes. The present invention relates to a method for efficiently extracting as an active ingredient.

  In recent years, one of the triggers is satiety due to an abundant lifestyle, westernization of eating habits due to high-fat diet intake, a decrease in physical labor due to changes in the social environment, a corresponding decrease in exercise, and stress. Due to heavy drinking and eating, modern people are prone to obesity.

  Obesity continues to increase, and WHO (World Health Organization) warns countries around the world for increased risk for lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, arteriosclerosis, and fatty liver related to obesity Has been issued. Metabolic syndrome (visceral fat syndrome) is a condition in which multiple risk factors for arteriosclerosis such as abnormal glucose metabolism (diabetes), abnormal lipid metabolism (hyperlipidemia), and hypertension are caused by the accumulation of visceral fat due to obesity That's it.

  Metabolic syndrome is estimated to be 9.4 million in Japan in the 2004 National Health and Nutrition Survey, and the combined number of reserves is 19.6 million, especially from 40 to 74 years old. Most of them are middle-aged men, accounting for a quarter of them. From April 2008, medical insurers (National Health Insurance and Employee Insurance) will be conducting screening and health guidance business focusing on built-in fat type obesity for insured persons and dependents over 40 years old. The government is working on countermeasures such as being obliged.

  In metabolic syndrome, when diabetes is taken as an example, one of the causes is insulin resistance (abnormal glucose metabolism). Insulin resistance is a state in which the action effect of insulin is reduced. Insulin resistance is relatively common in obese people. When nutrients exceeding the basal metabolism are continuously taken up by cells, fat cells are enlarged and the secretion amount of a substance that improves the action of insulin (adiponectin) decreases, while the substance that inhibits the action of insulin (TNA-α and release) A lot of fatty acid) is secreted. As a result, the action effect of insulin decreases, and diabetes may eventually be caused. Thiazolidine derivatives are known as components of therapeutic agents that improve insulin resistance (for example, Patent Document 1). The mechanism of action of the therapeutic agent is that the derivative binds to PPARγ, which is a nuclear receptor, increases the activity of PPARγ, induces differentiation of preadipocytes into non-hypertrophic normal adipocytes, and simultaneously enlarges Induces adipocyte apoptosis. Differentiated fat cells actively secrete adiponectin, so that sugar and lipid metabolism become active, sugar uptake from blood is promoted, and insulin resistance is improved. Such a function of fat cells leads to prevention of not only diabetes but also hyperlipidemia. Such an effect has also been confirmed with saponins contained in medicinal carrots.

  However, as described in Patent Document 1, when a synthetic compound is used, it may cause harmful side effects on the human body and is difficult to take on a daily basis like food. For this reason, the development of a substance that exhibits an excellent differentiation promoting effect without causing harmful side effects on the human body is awaited.

  In view of these problems, research has been conducted on food-derived extracts that can be taken orally and have the effect of promoting differentiation into adipocytes. For example, a composition derived from a sprout of a cruciferous plant (for example, Patent Document 2), at least one amino acid having a glucagon secretion enhancing action, at least one xanthine derivative, at least one isoflavone, and carnitines or A composition for preventing, improving or treating metabolic syndrome containing at least one kind of carnitine precursor (for example, Patent Document 3) has been reported. The composition of Patent Document 2 has cell differentiation promoting activity from preadipocytes to adipocytes, and can be expected to prevent or improve the progression of diabetes and related diseases. Moreover, the composition of patent document 3 has the effect | action which suppresses the synthesis | combination of fat in the living body, accelerates | stimulates decomposition | disassembly of fat, and burns fat effectively.

JP-A-8-143556 JP 2007-70271 A JP 2008-291002 A

  The present inventors have extensively screened the activity of promoting the differentiation of preadipocytes into adipocytes for food materials that can be taken orally as described above. As a result, it was found that the mushroom extract has the ability to activate PPARγ and promotes the differentiation of preadipocytes into adipocytes. At this time, conventionally, a mushroom extract having PPARγ activation ability has been obtained by water extraction, particularly hot water extraction.

  However, in this method, the yield (especially extraction efficiency) of the target active ingredient from mushrooms was not sufficient. For this reason, a method for efficiently extracting a target active ingredient from mushrooms has been desired.

  That is, an object of the present invention is to provide a method for efficiently extracting an active ingredient that activates PPARγ, a nuclear receptor, from mushrooms and promotes differentiation from preadipocytes to adipocytes.

  The present inventors have intensively studied to achieve the above object. As a result, combining conventional hot water extraction with heating (boiling) operation that also serves as sterilization (pre-sterilization) and enzyme treatment operation that causes cellulase or hemicellulase, which is a plant tissue disrupting enzyme, to act at a predetermined pH, The inventors have found that the extraction efficiency can be increased and completed the present invention.

  According to the method of the present invention, an active ingredient having an action of promoting differentiation from preadipocytes to adipocytes can be efficiently produced from mushrooms.

6 is a graph showing the results of a comprehensive transition of a fasting blood glucose level measurement test in the human monitor test of Example 2. 6 is a graph showing the results of changes for each sample in a fasting blood glucose level measurement test in the human monitor test of Example 2. It is a graph which shows the result of the comprehensive transition about the group of 150 mg / dL or more and less than 300 mg / dL among the fasting neutral fat measurement tests in the human monitor test of Example 2. It is a graph which shows the result of the comprehensive transition about the group of 300 mg / dL or more among the measurement tests of fasting neutral fat in the human monitor test of Example 2. It is a graph which shows the result of the transition for every sample about the group of 150 mg / dL or more and less than 300 mg / dL among the fasting neutral fat measurement tests in the human monitor test of Example 2. It is a graph which shows the transition result for every sample about the group of 300 mg / dL or more among the measurement tests of fasting neutral fat in the human monitor test of Example 2.

  The present invention relates to a method for efficiently producing (extracting) an active ingredient that promotes differentiation of adipocytes from mushrooms. In the present specification, “active ingredient” and “adipocyte differentiation promoting substance” are used interchangeably. Specifically, (1) After adding water 50 to 50 times the mass of the mushroom to the mushroom, the mushroom is crushed to obtain a crushed mushroom, and then the mashroom is heated. Sterilizing and partially decomposing the mushroom tissue to obtain a mushroom crushing heated product; (2) using the cellulase or hemicellulase at a temperature of 40-60 ° C. (3) heating the mushroom-crushed enzyme-treated product to 40 to 121 ° C. and performing hot water extraction to obtain a mushroom extract. In any of the steps (1) and (2), the acid of the mushroom crushed material is adjusted to 3 to 6. A method for producing a fat cell differentiation promoting agent which comprises adding. In the present specification, “crushed mushroom” includes mushroom, mushroom crushed material, mushroom crushed heated product in step (1), and mushroom crushed enzyme treated product obtained in step (2). Used with intention. That is, the to-be-treated product to which the acid is added so as to have a pH of 3 to 6 is a crushed mushroom.

  According to such a method, the extraction efficiency (yield) of the target active ingredient from the mushrooms can be increased.

  Moreover, the active ingredient manufactured by the said method has high PPARγ activation ability, and is excellent in the activity which promotes differentiation of preadipocytes into adipocytes. Here, “PPAR (peroxisome proliferator activated receptor)” is a nuclear receptor protein belonging to the steroid receptor superfamily, and three subtypes of α, γ, and δ have been reported so far. Yes. Among these, PPARα is highly expressed in the liver, pancreas, and skeletal muscle and regulates fatty acid combustion, and PPARγ plays an important role in adipocyte differentiation. When the activity of PPARγ is increased, differentiation of preadipocytes into normal fat cells that are not enlarged is induced, and at the same time, apoptosis of enlarged fat cells is induced. Differentiated fat cells actively secrete adiponectin, so that sugar and lipid metabolism become active, sugar uptake from blood is promoted, and insulin resistance is improved. Such a function of fat cells leads to prevention of not only diabetes but also hyperlipidemia.

  Therefore, the adipocyte differentiation promoting substance as an active ingredient produced by the method of the present invention is a pharmaceutical, food (health food), beverage (health drink), etc. for the purpose of promoting cell differentiation from preadipocytes to adipocytes. You can expect to use it.

  Embodiments of the present invention will be described below.

1. Process (1)
In this step, water having a mass of 0.5 to 50 times the mass of the mushroom is added to the mushroom, and then the mushroom is crushed to obtain a crushed mushroom.

  The mushroom used in the present invention is expressed as Agaricus bisporus or Agaricus campestris belonging to the genus Agaricaceae (Agaricus) in terms of biological classification.

  There are no particular limitations on the variety, and examples include white, off-white, cream, brown, and Agaricus bitorquis, and any of these can be preferably used.

  Further, the usable part of the mushroom is not particularly limited, and one or more kinds selected from a part made of an umbrella, a fold, a hole, a handle, a meat, a brim, a pot, a stone butt, a greba, and the like can be mentioned. It is done. That is, a partial position may be used alone, or a plurality of sites may be mixed and used.

  Further, the mushroom is preferably used regardless of whether it is raw, dried, frozen, or freeze-dried.

  Although the mushroom to be used can be used as it is, it is preferably washed with water. By washing with water, impurities attached to the mushroom can be removed, and the purity of the extract can be improved. In such a case, the mushroom may be dried after washing the mushroom with water.

  In this step, 0.5 to 50 times the mass of water is added to the mushroom and crushed to obtain a crushed mushroom. The amount of water added is not particularly limited as long as it is within the above range, but it is preferable to adjust appropriately depending on the state of the mushroom. For example, when the mushroom is used in a raw or frozen state, the mushroom contains some water. For this reason, in such a case, it is sufficient to add a relatively small amount of water. Specifically, the water is 0.5 to 10 times the mass of the mushroom, more preferably It is preferable to add 1 to 5 times the mass. Further, when the mushroom is used in the form of a dried product or a lyophilized product, the mushroom contains little or little water. For this reason, in such a case, it is necessary to add water in a relatively large amount as compared with the case of using it in a raw or frozen state. Specifically, the water is added to the mass of the mushroom. It is preferable to add 5 to 50 times the mass, more preferably 10 to 30 times the mass. If it is such a range, a mushroom can be crushed efficiently. In the present specification, “relative to the mass of the mushroom” means that the mass of the mushroom that is the starting material in the step (1) is used as a reference.

  Moreover, the water used in this step is not particularly limited, and any of tap water, distilled water, pure water, ion exchange water, and the like may be used.

  Moreover, the crushing of this process is not specifically limited, It is performed by a well-known method. For example, a crusher such as a blender, a stone mill type crusher, a roller mill, a cutter mill, a ball mill, a jet mill, a hammer mill or the like can be used. Alternatively, after the mushroom is mechanically processed in advance using a pulverizer as described above to obtain a coarse piece of mushroom, the coarse piece is further crushed by ultrasonic waves to obtain a crushed mushroom. Good. In the latter case, the mushroom that is mechanically processed is preferably crushed so as to have a size of about 10 μm or less. The ultrasonic crushing conditions are not particularly limited as long as the mushrooms have an appropriate size, but the treatment is preferably performed for 5 to 20 minutes. The size of the mushroom crushed material obtained in this step is not particularly limited. Preferably, the size of the crushed mushroom is about 0.001 to 2 mm, more preferably about 0.005 to 1 mm. If it is such a magnitude | size, an enzyme process will be efficiently performed by the following process (2), and the improvement of the extraction efficiency of an active ingredient by the hot water extraction operation in a process (3) can be anticipated.

  In the above operation, if necessary, water may be added after crushing for the purpose of improving the efficiency of heat treatment or enzyme treatment. At this time, the amount of water added is not particularly limited, and is appropriately selected depending on the desired effect. Preferably, in step (1), the total amount of water added to the mushroom is such an amount that is in the above range relative to the mass of the mushroom.

  Next, the obtained mushroom crushed material is heated (sterilized). Since many microorganisms adhere to the mushroom, if the sterilization treatment is not performed, these microorganisms will grow in the next step (2), and the microorganisms themselves or secretions from the microorganisms will inhibit the enzyme treatment in the next step. there's a possibility that. In addition, since the microorganisms propagated in the next step (2) or (3) may assimilate the extracted components or secrete toxins that are not preferable as food, such heating (sterilization) operation, Propagation of bacteria attached to the mushroom can be prevented in the subsequent manufacturing process. In addition, since the tissue (for example, cell wall) of the mushroom crushed material can be further partially pyrolyzed, the obtained mushroom crushed heated material is easily subjected to an enzyme treatment in the next step (2). The extraction efficiency in (3) can be increased.

  The heating conditions for the crushed mushroom are not particularly limited as long as the above-described effects can be achieved. For example, the crushed mushroom is preferably heated at 80 to 121 ° C. for 5 to 60 minutes, and more preferably heated at 80 to 121 ° C. for 10 to 30 minutes.

2. Process (2)
In this step, the mushroom crushing heated product obtained in the above step (1) is cooled to about 40 to 60 ° C., and then cellulase or hemicellulase is added to perform enzyme treatment to obtain a mushroom crushing enzyme treated product. By performing the enzyme treatment, it becomes possible to decompose the cell wall of the mushroom, efficiently release the active ingredient from the cells, and improve the extraction efficiency in the next step (3).

  Here, cellulase or hemicellulase is a plant tissue disrupting enzyme. Cellulase and hemicellulase may be used alone or in combination. Here, cellulase and hemicellulase may be derived from any source having cellulase activity or hemicellulase activity, and are not particularly limited. For example, aspergillus niger, Trichoderma reesei, Trichoderma viride, Trichoderma longibrachiatum, Streptomyces lividans, Bacillus subtilis, Pyrococcus horikoshii, Clostridium thermocellum, Humicola, Pyrococcus horikoshii, etc .; Abalone, Ezobora Derived from shellfish such as bivalves; Derived from nematodes such as pine wood nematodes; Etc. Cellulase and hemicellulase may be produced from the above-mentioned appropriate sources by known methods, but commercially available products may be used. Examples of commercially available cellulases include cellulase A “Amano” 3, cellulase T “Amano” 4 (both manufactured by Amano Enzyme Co., Ltd.); Spezyme CP, GC220, Multifect CL, Prima First, Inage, Inage Neutra, Accelerase, Optimash, Multifect CX10L, Optimase CX40L, Puradax HA (all manufactured by Genencor Kyowa Co., Ltd.); GODO-TCF, GODO-TCL, GODO TCD-H3 (all manufactured by Godo Sake Co., Ltd.) ; Sofitergen C-1 (manufactured by Taisho Technos Co., Ltd.); super thermostable cellulase (manufactured by Thermostable Enzyme Laboratory Co., Ltd.); cell riser, cellulase XL-522, cell team C, cell riser HT Conch, cellulase SS , Rase XL-531 (all manufactured by Nagase ChemteX); Bakezyme XE (manufactured by Nippon Shibel Hegner); Cellsoft, Denimax, Carezyme, Cellzyme, Cellcrust (all manufactured by Novozymes Japan) ); Cellulosin AC40, cellulosin AL, cellulosin T3, cellulosin TF (A feed) (all manufactured by HIBI Co., Ltd.); cellulase "Onozuka" 3S, cellulase Y-NC, pancerase br (all manufactured by Yakult Pharmaceutical Co., Ltd.) CellSEB Ts (manufactured by Higuchi Shokai Co., Ltd.); Sumiteam AC, Sumiteam C (both manufactured by Shin Nippon Chemical Industry Co., Ltd.); Sucrase C (Mitsubishi Chemical Foods Co., Ltd.); Entilon CM, Entilon MCH, Bio Hits, biostars, fedolases (any Rakuto Kasei Kogyo Co., Ltd.), and the like. Commercially available hemicellulases include, for example, hemicellulase “Amano” 90 (manufactured by Amano Enzyme Co., Ltd.); Bakezyme HS2000, Bakezyme 1 Conc (both manufactured by Nihon Shibel Hegner Co., Ltd.); Enchiron LQ (Santo Kasei) Kogyo Co., Ltd.). The cellulase or hemicellulase is not limited to the above. Moreover, the cellulase or hemicellulase may be used alone or in combination of two or more kinds, or a combination of cellulase and hemicellulase may be used as appropriate.

  Further, the amount of cellulase or hemicellulase added is not particularly limited as long as it can sufficiently treat the heated mushrooms obtained in the step (1), and is appropriately selected depending on the type of enzyme used. it can. For example, the addition amount of cellulase or hemicellulase is preferably 0.01 to 10% by mass, and 0.02 to 5% by mass with respect to the mass of the mushroom which is the starting material in step (1). Is more preferable. If it is such a range, a mushroom crushing heating material can fully be processed with an enzyme. In addition, it is preferable to mix with a stirrer etc. so that a mushroom crushing heating material and an enzyme (cellulase and hemicellulase) can mix uniformly and fully during an enzyme process.

  The enzyme treatment conditions are also not particularly limited as long as the mushroom crushed and heated product obtained in the step (1) can be sufficiently treated with the enzyme. For example, the enzyme treatment temperature is preferably 30 to 60 ° C., more preferably 35 to 55 ° C., and the enzyme treatment time is preferably 0.1 to 16 hours, more preferably 1 to 8 hours. In addition, when using 2 or more types of enzymes together, you may add simultaneously and perform an enzyme treatment, or you may repeat an enzyme treatment using each enzyme independently. Of these, when the optimum conditions for each enzyme are different, the latter treatment is preferably performed. In addition, when performing an enzyme process repeatedly, it is preferable to perform an enzyme deactivation process between each enzyme process.

  In this invention, an acid is added so that the pH of a mushroom crushed material or a mushroom crushed heated product may be 3 to 6 in either of the steps (1) or (2). In this specification, it may be simply described as “acid is added to the mushroom crushed material”.

  Thus, by adding acid, the pH of the mushroom, mushroom crushed product, mushroom crushed heated product or mushroom crushed enzyme-treated product is adjusted to a pH at which the enzyme easily acts, thereby further increasing the extraction efficiency of the active ingredient from the mushroom crushed heated product. Can be increased. Here, the timing of acid addition (pH adjustment) may be performed in either step (1) or step (2). Specifically, (a) acid is added to the mushroom before crushing; (b) acid is added to the mushroom crush before heating after crushing; (c) enzyme treatment after completion of step (1) The acid is added to the mushroom crushing heated product before; (d) the step (2) after the completion of the step (3), the acid is added to the mushroom crushing enzyme treatment product before the start, and the like. Said (a)-(d) may be implemented independently or may be implemented in combination of 2 or more type.

  In the said pH adjustment process, Preferably, an acid is added so that pH of a mushroom crushed material or a mushroom crushed heating material may be set to 3-6, More preferably, it is 3.5-5.5. In such a pH range, the activity of the enzyme is increased, and the extraction efficiency of the active ingredient from the mushroom crushed heated product can be further increased.

  Here, the acid is not particularly limited as long as it can increase the extraction efficiency of the active ingredient by adjusting the pH of the crushed mushroom or the heated mashroom to a predetermined level, and either an inorganic acid or an organic acid can be used. Can be used. Specifically, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, hydrofluoric acid; and malic acid, citric acid, tartaric acid, lactic acid, acetic acid, fumaric acid, phthalic acid, gluconic acid, adipic acid And organic acids such as vinegar, formic acid, oxalic acid, benzoic acid, and paratoluenesulfonic acid. Of these, organic acids are preferably used in consideration of safety, ease of pH adjustment, and the like. For example, when used for pharmaceuticals, foods (health foods) or beverages (health drinks), those that are recognized as food additives are preferable in consideration of safety and the like. Considering this point, hydrochloric acid, malic acid, citric acid, tartaric acid, lactic acid, acetic acid, fumaric acid, phthalic acid, gluconic acid, adipic acid, vinegar and the like that are recognized as food additives are preferable. Malic acid, citric acid, tartaric acid, lactic acid, acetic acid, fumaric acid, phthalic acid, gluconic acid, adipic acid, vinegar and the like are more preferable. In this case, the acid may be used alone or in the form of a mixture of two or more. The acid may be added as it is for the purpose of adjusting pH, but may be added in other forms such as an aqueous solution. In the latter case, the acid concentration in the solution is not particularly limited as long as the pH of the mushroom crushed product / mushroom crushed heated product can be adjusted to a predetermined level, and is appropriately adjusted depending on the ease of handling and the type of acid. Can be done.

3. Step (3)
In this step, the mushroom disrupted enzyme-treated product obtained in (2) above is heated to 40 to 121 ° C. to perform hot water extraction to obtain a mushroom extract. By this process, an active ingredient can be extracted with high efficiency from the mushroom disrupted enzyme-treated product.

  In this step, the hot water extraction conditions are not particularly limited as long as the active ingredients can be efficiently extracted from the mushroom disrupted enzyme-treated product. As described above, since water is added in step (1), the mushroom-crushed enzyme-treated product can be sufficiently extracted with hot water without adding water separately in this step. However, water may be added separately in this step, and the amount of water added in such a case is not particularly limited, but is usually 1.5 to 3 times the mass of the mushroom disrupted enzyme treatment product. The mass of water can also be added.

  Moreover, hot water extraction temperature is 40-121 degreeC, Preferably it is 40-90 degreeC. Here, when the hot water extraction temperature is less than 40 ° C., the temperature of the water is too low and the extraction efficiency is not sufficient. Conversely, when it exceeds 121 ° C., the temperature is too high and the active ingredient is decomposed. there is a possibility. The hot water extraction time is preferably in the range of 1 to 16 hours, more preferably 1 to 8 hours. During the extraction operation, the mushroom disrupted enzyme-treated product may be stirred. By stirring, heat is evenly distributed to the mushroom disrupted enzyme-treated product, and hot water extraction can be performed evenly. In addition, although the said extraction process may be performed once, when extraction efficiency is low, you may perform repeatedly twice or more.

  The method of the present invention includes the steps (1) to (3), but after the step (3), the mushroom extract obtained in the step (3) is further centrifuged or filtered to separate the residue. Thus, it is preferable to obtain an extract (step (4)). Thereby, a water-insoluble part is separated into solid and liquid. Here, in order to separate the residue, centrifugation or filtration is performed, but both centrifugation and filtration may be performed.

4). Process (4)
Centrifugation conditions are not particularly limited as long as water-insoluble portions can be separated. Specifically, the mushroom extract is preferably centrifuged at 4 to 50 ° C. at 3000 to 12000 rpm for 5 to 30 minutes, and centrifuged at 4 to 30 ° C. at 4000 to 10,000 rpm for 5 minutes to 30 minutes. More preferably.

  The filtration operation is not particularly limited as long as the water-insoluble part can be separated, and a known method can be adopted. For example, filter paper, microfilter, etc. are used. The pore size of the filter is not particularly limited as long as the water-insoluble portion can be separated while leaving the active ingredient. Specifically, the pore size of the filter is preferably 0.1 to 5 μm, more preferably 0.2 to 3 μm.

  In addition, the said centrifugation or filtration operation may each be performed once or may be repeated 2 times or more, or may be performed combining the said centrifugation and filtration operation suitably. In the case where the operation is repeated, the operation conditions may be the same or different.

  After the step (3), it is preferable that the extract obtained in (4) is further concentrated to obtain a concentrated solution (step (5)).

5. Process (5)
The concentration method is not particularly limited, and known methods such as vacuum concentration, ultrafiltration, vacuum concentration, freeze concentration, membrane concentration and the like can be used. The concentration conditions are not particularly limited, but are usually 80 ° C. or lower, more preferably 30 to 70 ° C., and it is preferable to concentrate under reduced pressure using a concentrator. Further, the amount of the liquid after concentration is not particularly limited, and may be an appropriate mass depending on the situation of use.

  After the step (5), the concentrated solution is preferably sterilized at 80 ° C. or higher to obtain a sterilized extract (step (6)). By such an operation, safety is ensured and it is suitably used for pharmaceuticals, foods (health foods) or beverages (health drinks).

6). Step (6)
If the sterilization condition is 80 ° C. or higher, the concentrated solution can be sufficiently sterilized, but preferably at a temperature of 80 to 121 ° C., more preferably 90 to 121 ° C., preferably 10 to 60 minutes, more preferably 10 to 30. Sterilize the concentrate for minutes. If it is such temperature and time range, it can fully sterilize, maintaining the activity of the active ingredient in a concentrate. The sterilization operation may be performed once or repeated two or more times. When the sterilization operation is repeated, the operation conditions may be the same or different. .

  After the step (6), it is preferable to further dry the sterilized extract obtained in the step (6) (step (7)). The dry powder obtained by the step (7) is excellent in storage (storage) stability because of the absence of water, and is light in mass, so it is convenient for transportation.

7). Step (7)
The method for drying the concentrate is not particularly limited, and a known method can be used. For example, freeze drying, spray drying drying, hot air drying, vacuum drying, steam drying, barrel drying, spin drying, suction drying, infrared drying, convection heat transfer drying, airflow drying, fluidized bed drying, superheated steam drying, rotating drum drying, The concentrated liquid can also be made into a dry powder by a method such as microwave drying or supercritical drying.

  According to the method of the present invention, high extraction efficiency can be achieved, so that the intended active ingredient can be obtained in high yield while maintaining its activity. Moreover, the active ingredient manufactured according to the method of the present invention has a PPARγ activation ability and has a preadipocyte differentiation induction promoting action (adipocyte differentiation promoting action). For this reason, the active ingredient manufactured according to the method of the present invention can be expected to be used for the prevention and treatment of diabetes and the prevention and treatment of hyperlipidemia. That is, the active ingredient produced according to the method of the present invention induces apoptosis of enlarged adipocytes by promoting differentiation from preadipocytes to normal adipocytes. Differentiated fat cells actively secrete adiponectin, so that sugar and lipid metabolism become active, sugar uptake from blood is promoted, and insulin resistance is improved. Such a function of fat cells leads to prevention of not only diabetes but also hyperlipidemia.

  Furthermore, since the above-described method of the present invention does not use solvents or conditions that are problematic in food safety, the obtained active ingredient (adipocyte differentiation promoting substance) is a pharmaceutical, food (health food) Or it can be used in a beverage (a health beverage as it is (concentrated and dried powder)) or by appropriately processing the above-mentioned form.

  That is, the active ingredient (adipocyte differentiation promoting substance) obtained by the method of the present invention becomes an active ingredient of a pharmaceutical, food (health food) or beverage (health drink), and is appropriately prepared by drying, concentration or dilution, In some cases, by further purification, pharmaceuticals and foods having various dosage forms and forms can be obtained. Hereinafter, the pharmaceutical and food according to this embodiment will be described.

  The pharmaceutical of this embodiment is not particularly limited as long as it is a prophylactic or therapeutic agent for diseases caused by insulin resistance, and includes, for example, a prophylactic or therapeutic agent for diabetes, hypertension, arteriosclerosis and the like. It is done.

  The pharmaceutical dosage form can be tablets, pills, powders, powders, granules, syrups, solutions, suspensions, emulsions or capsules, and these can be administered orally to patients. The pharmaceutical may be administered transdermally as an external preparation such as an ointment, cream, powdery or liquid coating agent, or patch. A preferable dosage form or administration form of the pharmaceutical product according to this embodiment is appropriately selected by a doctor according to the age, sex, constitution, symptom, treatment timing, etc. of the patient.

  On the other hand, semi-solid preparations such as lotions, creams and ointments of the above-mentioned pharmaceuticals are prepared by converting the above-mentioned pharmaceuticals into fats, fatty oils, lanolin, petrolatum, paraffin, wax, plasters, resins, plastics, glycols, higher alcohols, glycerin. , Water, an emulsifier, a suspending agent, and the like.

  The content of the active ingredient (adipocyte differentiation promoting substance) according to the present invention contained in the medicinal product varies depending on the extraction method / condition, dosage form, severity, desired dose, etc. The total mass is preferably 0.01 to 30% by mass, more preferably 0.05 to 20% by mass in terms of dry matter.

  The dosage of the pharmaceutical agent varies depending on the age, weight and symptoms of the patient, the intended mode and method of administration, therapeutic effect, treatment period, etc., and the exact amount is determined by a doctor. For example, when the drug is orally administered, 0.05 to 1,000 mg per day for an adult in terms of the dose (dry matter equivalent) of the active ingredient (adipocyte differentiation promoting substance) Can be administered in one or several divided doses.

  Next, the type of food of this embodiment is not particularly limited. That is, the active ingredient (adipocyte differentiation promoting substance) according to the present invention has an adipocyte differentiation promoting action that can improve insulin resistance and significantly reduce the risk of causing side effects such as liver dysfunction. Have. Thus, the dietary diet of this embodiment can play an important role in improving insulin resistance and regulating hyperinsulinemia. Furthermore, the food may contribute to the prevention of metabolic syndrome disease for humans who are insulin resistant and who will suffer from metabolic syndrome in the future.

  The content of the active ingredient (adipocyte differentiation promoting substance) according to the present invention contained in the food is not particularly limited because it varies depending on the composition of each food, but with respect to the total mass of the food In terms of dry matter, the content is preferably 0.01 to 30% by mass, and more preferably 0.05 to 20% by mass. When it is within the above-described range, the effects of the above-described diet therapy can be significantly improved.

  Specific examples of the food include salmon, chewing gum, milk, yogurt, whey beverage, lactic acid bacteria beverage, juice, tea, beverage, ice cream, pudding, water candy and the like. Although the said foodstuff is not limited to the following, what is used as a normal food ingredient for the said active ingredient, ie, dextrin, cellulose, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside , Corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose Fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B, nicotinamide, calcium pantothenate, amino acids, calcium Salts, dyes, can also be obtained by suitably blending at least one selected from the group consisting of fragrances and preservatives.

  Next, the type of beverage of this aspect is not particularly limited. That is, the active ingredient (adipocyte differentiation promoting substance) according to the present invention has an adipocyte differentiation promoting action that can improve insulin resistance and significantly reduce the risk of causing side effects such as liver dysfunction. Have. Therefore, the diet therapy with the beverage of this embodiment can play an important role in improving insulin resistance and regulating hyperinsulinemia, similar to the diet therapy with the food. Furthermore, the food may contribute to the prevention of metabolic syndrome disease for humans who are insulin resistant and who will suffer from metabolic syndrome in the future.

  The content of the active ingredient (adipocyte differentiation promoting substance) according to the present invention contained in the beverage is not particularly limited because it varies depending on the composition of each beverage, but with respect to the total mass of the beverage In terms of dry matter, the content is preferably 0.01 to 30% by mass, and more preferably 0.05 to 20% by mass. When it is within the above-described range, the effects of the above-described diet therapy can be significantly improved.

  Specific examples of the beverage include juice, soft drink, tea, coffee, tea, carbonated drink, sports drink, soup, and nutrition drink. In addition to the active ingredient (adipocyte differentiation promoting substance) according to the present invention, the beverage may contain other components similar to those in the past, and these other components are not particularly limited, It is the same as a well-known component.

  It should be noted that the scope of the present invention is not limited to the above-described embodiments, and those skilled in the art can make various modifications without departing from the spirit of the present invention.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

Example 1
165 g of commercially available raw mushrooms were weighed and washed with water, 165 ml of an equal amount of distilled water was added and crushed with a blender to obtain a crushed mushroom having a size of about 1 mm or less. The obtained mushroom crushed product was boiled at 100 ° C. for 10 minutes for the purpose of partially decomposing the cell wall of the mushroom crushed product and sterilizing to obtain a mushroom crushed heated product. Next, 330 g of distilled water was added to the heated mushroom product. Then, 1.05 mass% citric acid was added with respect to the mass of a raw mushroom, pH was adjusted to 4, and it cooled to 40 degreeC. Furthermore, the cellulase preparation which is a plant tissue disintegrating enzyme of 0.1% by mass with respect to the mass of the raw mushroom is added to the heated product of crushing mushroom, and the enzyme treatment is performed at 40 ° C. for 1 hour while gently stirring. To obtain a mushroom-crushed enzyme-treated product.

  Next, this mushroom-crushed enzyme-treated product was heated to 60 ° C. and subjected to hot water extraction for 2 hours while gently stirring to obtain a mushroom extract. The obtained mushroom extract was centrifuged at 8000 rpm for 10 minutes, and then filtered through a filter paper to remove the residue to obtain an extract. The extract was concentrated under reduced pressure using an evaporator to obtain a concentrated solution. Then, it boiled at 100 degreeC for 10 minute (s), sterilized, and obtained the sterilized extract. The obtained sterilized extract was freeze-dried to obtain about 11.1 g of a dry powder.

Comparative Example 1
150 g of commercially available raw mushrooms were weighed and washed with water, and an equal amount of distilled water 150 g was added and crushed with a blender to obtain a crushed mushroom having a size of about 1 mm or less. 300 g of distilled water was added to the obtained mushroom crushed material.

  Next, this mushroom crushed material was heated to 60 ° C. and subjected to hot water extraction for 2 hours while gently stirring to obtain a mushroom extract. The obtained mushroom extract was centrifuged at 8000 rpm for 10 minutes, and then filtered through a filter paper to remove the residue to obtain an extract. The extract was concentrated under reduced pressure using an evaporator to obtain a concentrated solution. The obtained concentrated liquid was freeze-dried to obtain about 8.14 g of a dry powder.

Example 2: Evaluation of PPARγ activation ability of mushroom extract In this example, the dry powder prepared in Example 1 and Comparative Example 1 was subjected to PPARγ activation ability as adipocyte differentiation induction promoting activity according to the following method. Was measured.

1. Preparation method of test sample Each of the dry powders prepared in Example 1 and Comparative Example 1 was dissolved in sterile water to a concentration of 100 mg / ml (w / v solution) to prepare a test sample.

  In addition, troglitazone was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 μM to prepare a troglitazone solution.

2. Transformation of COS-1 cells and addition of test sample COS-1 cells were collected by trypsin treatment, centrifuged at 1000 rpm and 4 ° C for 3 minutes, and then the supernatant was removed. The obtained cells were dispersed in 2 ml of a medium, seeded in a 60 mm culture dish (Corning) at a density of 5 × 10 ⁵ cells / well, and then at 37 ° C. in the presence of 5% CO _{2 for} 24 hours. And cultured.

For transformation, Effectene Transfection Reagent (QIAGEN) was used. That is, Buffer EC 150 μl, pM-hPPARγ-GAL4 1 μg, pUASg-tk-Luc 1 μg, pSEAP-control vector 1 μg, and finally Enhancer 24 μl were placed in a 1.5 ml tube and vortexed for 1 second. . After leaving at 25 ° C. for 3 minutes, 25 μl of Effectene was added, stirred for 10 seconds with vortex, and then left at 25 ° C. for 7 minutes. During this time, the medium of the 60 mm culture dish was removed, and 4 ml of fresh medium was added to replace the medium. After 7 minutes, 1 ml of the medium was added to a 1.5 ml tube, suspended twice by pipetting, the whole amount was dropped into a 60 mm culture dish, and cultured for 16 hours in the presence of 37 ° C. and 5% CO ₂ . .

The transformed cells were collected by trypsin treatment, centrifuged at 1000 rpm and 4 ° C. for 3 minutes, and then the supernatant was removed. The obtained cells were suspended in 10 ml of a medium and seeded on a 96-well plate (NUNC) at 125 μl / well and cultured at 37 ° C. in the presence of 5% CO _{2 for} 1 to 2 hours. Thereafter, the above 1. The test sample prepared in (1) was added to 1.25 μl / well, gently agitated, and cultured in the presence of 37 ° C. and 5% CO _{2 for} 24 hours.

3. Measurement of Luciferase activity 2. In 24, 24 hours after the addition of the test sample, 25 μl / well of the medium was collected from the 96-well plate and transferred to the 96-well white plate. Thereafter, 100 μl / well of a luciferase activity measurement solution melted at 37 ° C. was added to the remaining 100 μl / well, reacted for 35 minutes in the dark, and then the luminescence intensity of each Luciferase (the following formula) Among them, “Luciferase luminescence intensity of the extract”) was measured. In the above method, as a comparative control, a similar experiment was performed except that sterilized water was added instead of adding the test sample (“Luciferase luminescence intensity of sterilized water” in the following formula). Similarly, in the above method, instead of adding the test sample as a comparison control, the above 1. The same experiment was performed except that the troglizonezone solution and DMSO prepared in (1) were added (“Troglizone luciferase luminescence intensity” and “DMSO luciferase luminescence intensity” in the following formulas, respectively). Moreover, the solution for measuring Luciferase activity is the following 5. Prepared according to

4). Measurement of secreted alkaline phosphatase (SEAP) activity In 24, 24 hours after addition of the test sample, 1 × Dilution Buffer 25 μl / well was added to 25 μl / well of the medium collected from the 96-well plate. After capping with cellophane tape, the mixture was gently stirred and left at 65 ° C. for 30 minutes. Then, after cooling to 4 degreeC and returning to 25 degreeC, Assay Buffer 90microliter / well was added and it stirred gently. The mixture was left at 25 ° C. for 5 minutes, 10 μl / well of MUP solution was added, and the mixture was gently stirred. After reacting at 25 ° C. for 60 minutes in the dark, the fluorescence intensity of SEAP (Ex = 360 nm, Em = 460 nm) (“SEAP fluorescence intensity of the extract” in the following formula) is measured based on 4-methyl umbelliferone. did. In the above method, as a comparative control, a similar experiment was performed except that sterilized water was added instead of adding the test sample (“SEAP fluorescence intensity of sterilized water” in the following formula). Similarly, in the above method, instead of adding the test sample as a comparison control, the above 1. The same experiment was performed except that the troglitazone solution and DMSO prepared in (1) were added (“Troglitazole SEAP fluorescence intensity” and “DMSO SEAP fluorescence intensity” in the following formulas, respectively). In addition, 1 × Dilution Buffer, Assay Buffer, and MUP solution are the following 5. Prepared according to

5. 2. Preparation of reagent The solution for measuring Luciferase activity used in 1 was prepared as follows. That is, 60 mM Tricine-NaOH (pH 7.8), 16 mM (MgCO ₃ ) ₄ Mg (OH) ₂ .5H ₂ O (basic MgCO ₃ ), 0.4 mM EDTA, 10% Surfact-Amps X-100 (Thermo) , 0.5 mM D-Luciferin potassium salt (Nacalai Tesque), 1.5 mM Adenosine 5′-triphosphate (SIGMA), 0.5 mM Coenzyme A (SIGMA), and 0.1 mM β-Mercaptethanol, About 10 ml was dispensed and stored at −20 ° C. until just before use.

The above 4. 1 × Dilution Buffer used in was prepared as follows. That is, immediately before use, 5 × Dilution Buffer was diluted with H ₂ O. Here, 5 × Dilution Buffer was prepared by dissolving 4.38 g of NaCl and 2.42 g of Tris in 90 ml of ultrapure water. 12N HCl was added to adjust to pH 7.2 and stored at 4 ° C. until just before use.

The above 4. The Assay Buffer used in 1 was prepared as follows. That is, 0.9 g of L-homoargine and 0.04 g of MgCl ₂ were dissolved in 158 ml of ultrapure water, and 42 ml of diethylamine were added. 12N HCl was added to adjust to pH 9.8 and stored at 4 ° C. until just before use.

  The above 4. The MUP solution used in was prepared as follows. That is, 1 × Dilution Buffer 2.7 μl / well, Assay Buffer 7 μl / well, and 10 × MUP 0.3 μl / well were mixed. 10 × MUP, 4-methylbelliferous phosphate (MUP) 2.56 mg was dissolved in 1000 μl of ultrapure water and stored at −20 ° C. until just before use.

6). Evaluation of PPARγ activation ability 3. 3. Luciferase luminescence intensity measured in 1 above, and 4. From the fluorescence intensity of SEAP measured in step 1, the relative luciferase activity / SEAP activity against troglizone was defined as the PPARγ activation ability, and was determined according to the following formula (1).

  In the above formula, (A) and (B) were calculated according to the following formulas (2) and (3).

  The yield, yield and PPARγ activation ability of Example 1 and Comparative Example 1 are summarized in Table 1 below.

  From the results shown in Table 1 above, the dry powder of Example 1 can achieve a higher yield and PPARγ activation ability than the dry powder of Comparative Example 1, and can efficiently extract active ingredients. It is shown that it can.

Example 3: Effect test of mushroom extract using human (clinical experiment)
<Method for preparing extract for human monitoring test>
70 kg of commercially available raw mushrooms were weighed and washed, and an equal amount of 70 kg of tap water was added and crushed with a mascolloider to obtain crushed mushrooms. 140 kg of tap water was added to the obtained mushroom crushed material. Thereafter, 735 g of citric acid was added to make the pH 1.0, so that the mass was 1.05% by mass with respect to the mass of the raw mushroom. Then, the cell wall of the mushroom crushed material was partially decomposed and heated at 85 ° C. for 10 minutes as a sterilization process to obtain a mushroom crushed heated product.

  Next, after the mushroom crushing heated product is cooled to 40 ° C., 70 g of cellulase preparation is suspended in 200 ml of tap water so that the mass of the raw mushroom is 0.1% by mass in the mushroom crushing heated product. Then, the enzyme treatment was performed while gently stirring at 40 ° C. for 1 hour to obtain a mushroom-crushed enzyme-treated product.

  Next, the mushroom disrupted enzyme-treated product was heated to 60 ° C., and hot water extraction was performed while gently stirring for 2 hours to obtain a mushroom extract.

  Then, after centrifuging with a multi-plate centrifuge, it filtered with a filter paper and Celite # 2000, and the residue was removed and the extract was obtained.

  The obtained extract was concentrated at 60 ° C. with a vacuum concentrator to obtain a concentrate. Thereafter, sterilization was performed at 110 ° C. for 5 minutes to obtain a sterilized extract. Then, Brix of the sterilized extract was measured, 30% dextrin was added to the solid conversion value, and 5.49 kg of dry powder of the extract was obtained by spray drying.

<Human monitor test method>
Table 2 below summarizes the test items and test contents.

  Furthermore, Table 3 shows the breakdown of the subject, and Table 4 shows evaluation items and the like.

<Measurement of fasting blood glucose level and results of its transition>
Fasting blood glucose levels were measured using the hexokinase UV method. At that time, JCA-BM series automatic analyzer cleaner (JCA-BM9020, JEOL Ltd.) was used as an instrument, and Quick Auto Neo GLU-HK (Sinotest) was used as a reagent.

  In the group (7 persons) whose fasting blood glucose level before the test was 100 mg / dL or higher, the blood glucose level before the test was 111.0 ± 14.1 mg / dL, whereas the blood glucose level after the test was 107. It became 0 ± 14.8 mg / dL. From these results, it was found that the blood glucose level after the test was about 96% as an average value compared with the blood glucose level before the test, and the blood glucose level was lowered. In addition, the result before and after the test of each individual and the whole is shown in Table 5 below (Glu (mg / dL)).

  Here, the results shown in Table 5 are shown in FIGS.

<Measurement of fasting triglyceride level and its transition>
Neutral fat (TG) was measured using an enzymatic method (GK-GPO · free glycerol). At that time, Hitachi 7600 was used as an instrument, and Pure Auto S TG-N (Sekisui Medical Co., Ltd.) was used as a reagent.

  In the group (8 subjects) whose fasting blood glucose level before the test was 150 mg / dL or more and less than 300 mg / dL, the triglyceride value before the test was 204.9 ± 42.0 mg / dL, whereas after the test The triglyceride value was 134.1 ± 43.1 mg / dL (p <0.01 vs test, t-test). On the other hand, the triglyceride value before the test was 430.3 ± 47.0 mg / dL in the group (three persons) of 300 mg / dL or more, whereas the triglyceride value after the test was 275 ± 60. It was 8 mg / dL. From these results, compared with the triglyceride value before the test, the triglyceride value after the test is about 65% (a group of 150 mg / dL or more and less than 300 mg / dL) and about 64% (300 mg), respectively. / DL or higher group), and it was found that the triglyceride value significantly decreased before and after the test. In addition, the result before and after each individual and the whole test is shown in Table 6 below (unit: mg / dL).

  Here, the results shown in Table 6 are shown in FIGS. 3A and 3B and FIGS. 4A and 4B. 3A and 4A show the results for the group of 150 mg / dL or more and less than 300 mg / dL, and FIGS. 3B and 4B show the results for the group of 300 mg / dL or more.

Claims (6)

(1) After adding 0.5 to 50 times the mass of water to the mushroom, the mushroom is crushed to obtain a crushed mushroom, and then the crushed mushroom is heated. Sterilizing and partially decomposing mushroom tissue to obtain a heated mushroom crushing material;
(2) A step of subjecting the heated mushroom disrupted product to an enzyme treatment using cellulase or hemicellulase at a temperature of 40 to 60 ° C. to obtain a mushroom disrupted enzyme treated product;
(3) The mushroom disrupted enzyme-treated product is heated to 40 to 121 ° C. to perform hot water extraction to obtain a mushroom extract,
A method for producing an adipocyte differentiation promoting substance, wherein an acid is added so that the pH of a crushed mushroom product is 3 to 6 in either of the steps (1) or (2).   The method according to claim 1, wherein in step (1), the heating is performed at 80 to 121 ° C. for 5 to 30 minutes.   The method according to claim 1, wherein the acid is an organic acid.   The method according to any one of claims 1 to 3, further comprising the step of (4) centrifuging or filtering the mushroom extract and separating the residue to obtain an extract after (3). Method.   The method according to claim 4, further comprising the step of (5) concentrating the extract to obtain a concentrate after the step (4).   6. The method according to claim 5, further comprising: (6) sterilizing the concentrated solution at 80 ° C. or higher to obtain a sterilized extract after the step (5); and (7) drying the sterilized extract. Method.

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