JP7205818B2 - Method for producing persimmon leaf extract - Google Patents

Description

TECHNICAL FIELD The present invention relates to a persimmon leaf extract, a method for producing the same, and the like.

Due to lifestyle factors such as the spread of high-calorie diets and the decrease in the amount of daily exercise, not only middle-aged and elderly people but also young people develop diabetes or exhibit insulin resistance. It is also known that aging of adipocytes causes insulin resistance, and the risk of developing diabetes is high even in elderly people who exercise less. In this way, the number of diabetes patients is increasing explosively year by year due to changes in daily life and the transition to an aging society. believed to exist. In this situation, since diabetes is a fundamentally irreversible disease, it is important to implement measures that do not aggravate the condition of diabetic patients, and prevent the development of diabetes in people with insulin resistance. is important.

Dietary therapy, exercise therapy, and drug therapy are employed alone or in combination to address these issues. However, diet therapy that restricts calorie intake and exercise therapy that requires exercise for a certain period of time cannot be performed continuously in many cases. However, the decrease in the amount of exercise associated with a significant decrease in exercise capacity is also serious, and there are increasing cases where it is difficult to deal with it only by the above methods. Therefore, by focusing on the mechanism of uptake of sugar from blood vessels (blood) to skeletal muscle, investigations have been made to find solutions that are more effective than conventional methods, either alone or in combination with conventional therapies (Patent document 1, 2). A similar study was conducted on persimmon leaf extract, and a component was found that has the effect of taking in sugar from blood vessels (blood) to skeletal muscle (Patent Document 3). However, in subsequent studies, it is becoming clear that there are unknown ingredients in persimmon leaves that have the above-mentioned effects. There was a problem that the extract reflected in was not obtained.

JP 2010-37323 A JP 2011-37732 A JP 2014-198684 A

An object of the present invention is to provide an extract in which a component contained in persimmon leaves that has an action of taking in sugar from blood vessels (blood) to skeletal muscle is concentrated, and a method for producing the extract.

The present inventors have made intensive studies in view of such circumstances, and as a result, polyphenols that are present in persimmon leaf water or water-ethanol mixture extracts and that are adsorbed to styrene-divinylbenzene copolymers have been found to have a composition The persimmon leaf extract, which is characterized by containing 20% by mass or more based on the total amount of the composition and having a water content of 10% by mass or less based on the total amount of the composition, improves or enhances the glucose metabolism function in skeletal muscle. The present inventors have found that it has the effect of making them effective, and have made further improvements to complete the present invention.

That is, the present invention encompasses, for example, the subject matter described in the following sections.
Section 1. Contains 20% by mass or more of polyphenols adsorbed to styrene-divinylbenzene copolymer, which are present in persimmon leaf water or water-ethanol mixed liquid extract, and the water content is A persimmon leaf extract characterized by being 10% by mass or less relative to the total amount of the composition.
Section 2. Item 2. The persimmon leaf extract according to Item 1, wherein the total content of fructose, glucose and sucrose is 1.5% by mass or less relative to the total amount of the composition.
Item 3. Persimmon leaf water or water-ethanol mixture extract is adsorbed on an adsorbent containing a styrene-divinylbenzene copolymer resin, and a water-ethanol mixture containing 50 (vol/vol)% or more ethanol. A method for producing a persimmon leaf extract, comprising a step of eluting with a liquid.
Section 4. Item 1 or 2, food, medicine, or cosmetics using the persimmon leaf extract.
Item 5. Item 3. A composition for improving sugar metabolism, comprising the persimmon leaf extract according to Item 1 or 2.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain a persimmon leaf extract that has the effect of improving or improving the glucose metabolism function in skeletal muscle.

Fig. 3 shows the results of measurement of the sugar uptake-enhancing activity of a persimmon leaf extract not treated with resin adsorption or a persimmon leaf extract after resin adsorption treatment using skeletal myotube cells.

The present invention will be described in more detail below.

The persimmon leaf extract of the present invention contains a certain amount or more of polyphenols that are present in water or a water-ethanol mixture extract of persimmon leaves and adsorbs to styrene-divinylbenzene copolymer, and the water content is contains a persimmon leaf extract characterized by a certain amount or less.

The persimmon leaves used in the present invention are not particularly limited as long as they are leaves obtained from persimmons (Diospyros kaki L.), and those obtained from various varieties of persimmons can be used. For example, if it is a so-called sweet fruit variety, Fuyu, Jiro, Amahyakunichi, Gosho, Hanagosho, Bangosho, Tenjin Gosho, Fujiwara Gosho, Tokuda Gosho, Migatani Gosho, Zenjimaru, Tohachi, Mizushima, Shogatsu , etc., and the so-called astringent varieties include Yokono, Hiratanenashi, Fuji, Saijo, Dojo Hachiya, Aizumi Shirazu, Imon, Gionbo, Yotsumizo, Oyotsumizo, Atago, Hagakure, Examples include, but are not limited to, Kawabata, Takura, Sakushu Mishirazu, Nishimura, Fude, Tone Wase, and Koshu Hyakume. Persimmon leaves are preferably collected from early summer to autumn, more preferably from June to September. The collected persimmon leaves may be used for extraction as they are, but it is preferable to extract after drying. Drying methods include, for example, natural drying by leaving in a well-ventilated place, drying in the sun, drying by exposing to dried or heated gas, drying by exposing to high-temperature or high-pressure steam, and microwaves. Drying by irradiation, vacuum drying, freeze-drying, and the like can be used, but drying by exposure to a dried gas or a heated gas is preferable for hygienic reasons. Pretreatment may be performed when drying. For example, after collecting, it may be washed with water, cut into a suitable size (for example, a rectangular shape with a width of about 3 mm to 5 mm), and dried.

The method for extracting persimmon leaves is not particularly limited, and a known method such as immersion extraction can be used. In order to increase the extraction efficiency, a Soxhlet extraction method, a reflux extraction method, or the like can be used in addition to the immersion extraction method.

Water or a mixture of water and ethanol can be used as a solvent for extracting persimmon leaves. When a mixed solution of water and ethanol is used, it is preferable that the mixing ratio of water and ethanol be the same in terms of mass or that the content of water be high. Among them, it is most preferable to use water.

The amount of solvent (bath ratio) used for extracting the persimmon leaves can be about 10 to 50 in terms of mass of the extraction solvent per 1 persimmon leaf. Among them, 10 to 30 is preferable, and 15 to 25 is most preferable in consideration of extraction efficiency and concentration after extraction.

The extraction temperature is not particularly limited, but may be room temperature to about the boiling point of the solvent. When the extraction solvent is a mixture of water and ethanol, the extraction temperature is preferably 30°C to the boiling point, more preferably 60 to 80°C. When the extraction solvent is water, the extraction temperature is preferably 60-95°C, more preferably 85-95°C, most preferably 90-95°C. When extracting under temperature conditions lower than 40° C., about 5 to 15% by mass of ethanol or polyhydric alcohol can be blended for preservative purposes. Under extraction conditions below room temperature, this level of alcohol content does not interfere with extraction.

The extraction time can be appropriately determined based on the extraction conditions such as the temperature and stirring at the time of extraction. For example, it is usually preferably set within the range of 20 minutes to 24 hours, more preferably within the range of 25 minutes to 4 hours, and most preferably within the range of 30 minutes to 2 hours.

After the extraction is completed, solid-liquid separation is performed by appropriately combining filtration using filters such as metal mesh, filter cloth, filter paper, etc., and solid-liquid separation using mechanical force such as centrifugation and compression processing, to obtain an extract. can be done. In addition, after the extraction is completed, it may be concentrated.

The residue after the extraction treatment may be washed with a new extraction solvent to recover the extract liquid remaining in the residue. Moreover, the extraction process can be performed again with a new extraction solvent using the residue after the extraction process. Alternatively, liquids obtained by separate extraction can be combined.

The liquid extract obtained by the extraction process is purified and concentrated using an adsorbent. As the adsorbent, a styrene-divinylbenzene copolymer adsorption resin is used. In particular, an adsorption resin that efficiently adsorbs peptides and polyphenols having a molecular weight of 1000 daltons or more is preferable. The specific surface area of the adsorption resin is preferably 500-700 m ² /g. The mode radius of the adsorption resin is preferably 100-400 Å, more preferably 250-350 Å. Examples of adsorption resins include Amberlite XAD series (manufactured by Organo Corporation), Amberchrom CG series, Dowex Optipore series (manufactured by Dow Chemical Company), DIAION (registered trademark) HP series and SP series, MCI GEL CHP series, (manufactured by Mitsubishi Chemical Corporation), Serdolit series (manufactured by SERVA Electrophoresis GmbH), and the like.

After the extract is adsorbed on the adsorbent, it can be washed with water or a water-ethanol mixture, for example.

As a solvent for eluting the component adsorbed by the adsorbent, for example, a water-ethanol mixed solution can be used. The mixing ratio of water and ethanol is preferably the same amount in terms of mass or a higher ethanol content.

After elution, it may be further concentrated. The concentration method is not particularly limited, and can be carried out by a known method such as concentration under reduced pressure. Alternatively, it may be dried. The drying method is not particularly limited, and a known method such as drying under reduced pressure can be used.

The persimmon leaf extract of the present invention contains polyphenols, sugars, water and the like.

Examples of polyphenols contained in the persimmon leaf extract of the present invention include Kaempferol 3-O-galactoside (Trifolin) and Kaempferol 3-O-glucoside (Astragalin). The content of polyphenols is preferably 20% by mass or more, more preferably 30% by mass or more, relative to the total amount of the composition. The content of polyphenols is a value determined by the Folin-Ciocalteu method.

Sugars contained in the persimmon leaf extract of the present invention include fructose, glucose and sucrose. The total content of fructose, glucose and sucrose is preferably 1.5% by mass or less with respect to the total amount of the composition. More preferably, the contents of fructose, glucose, and sucrose are each 0.5% by mass or less. The sugar content is a value obtained by HPLC analysis under the following conditions.

[HPLC operating conditions]
Detector: RI
Column: Octadecylsilyl group-bonded silica gel packed column (ODS column) (Example: CAPCELL PAK NH2 UG80 S5)
Mobile phase: acetonitrile/water mixture (80:20)
Flow rate: 1.0 mL/min

The water content is preferably 10% by mass or less, more preferably 7% by mass or less, relative to the total amount of the composition. The water content is a value determined by the loss-on-drying method.

The persimmon leaf extract of the present invention can be used particularly preferably for improving sugar metabolism. Moreover, the persimmon leaf extract of the present invention is not particularly limited, but can be used, for example, as a raw material for foods, pharmaceuticals, cosmetics, and the like. In other words, the present invention includes a composition for improving sugar metabolism containing the persimmon leaf extract of the present invention. The present invention also includes foods, pharmaceuticals, cosmetics and the like using the persimmon leaf extract of the present invention. Furthermore, since these foods, medicines, cosmetics and the like contain the persimmon leaf extract of the present invention, they can be preferably used for improving sugar metabolism.

The composition for improving sugar metabolism of the present invention may be the persimmon leaf extract itself, or may contain other components in addition to the persimmon leaf extract. Other components include pharmaceutically or food hygienically acceptable carriers.

Moreover, the composition for improving glucose metabolism of the present invention can be used as an oral composition. Oral compositions include pharmaceuticals, quasi-drugs, diabetes, hyperglycemia, glucose intolerance, insulin resistance, arteriosclerosis, obesity, body fat accumulation, dyslipidemia, lifestyle-related diseases, prevention of fatty liver, etc.・Foods such as functional foods and drinks with the concept of improving, enhancing sugar metabolism, and anti-aging physiological functions, foods for sick people, foods for specified health use, foods with nutrient function claims, nutritional supplements, foods for exercise therapy, and foods for slimming. can be

The form of the composition for improving sugar metabolism of the present invention is not particularly limited, but when it is used as an oral composition, for example, forms such as hard capsules, soft capsules, supplements, chewable tablets, beverages, powdered beverages, granules, films, etc. can be Also, for example, tea drinks, sports drinks, beauty drinks, fruit juice drinks, carbonated drinks, alcoholic drinks, soft drinks, jelly drinks, water, hot water, beverages such as concentrated drinks diluted with carbonated water, etc. Powders and granules that are dissolved or suspended in liquids, dry solids such as tablets, tablets, jellies, snacks, baked goods, fried goods, cakes, chocolates, gums, candies, gummies and other confectionery , soups, noodles, cooked rice, cereals and the like. Among these, forms such as supplement type, chewable tablet, and one-shot drink type are preferable when used in normal life, and beverage forms such as sports drinks are most preferable when ingested for the purpose of enhancing exercise effects. . Additionally, these oral compositions can be provided to the consumer as packaged foods in containers. The container is not particularly limited as long as it can be sealed.

In the case of oral solid preparations such as tablets, granules and capsules, oral liquid preparations such as oral liquid preparations and syrup preparations, the total amount of the oral composition in terms of dry matter is, for example, the persimmon leaf of the present invention. About 0.01 to 95% by mass, preferably about 5 to 90% by mass, and most preferably about 10 to 80% by mass of the extract can be blended. In the case of an oral composition other than the above, for example, the persimmon leaf extract of the present invention is added in an amount of about 0.001 to 50% by mass, preferably about 0.005 to 30% by mass, most preferably 0.01 to 10% by mass. can be blended to some extent.

Production Example 1: Production of Persimmon Leaf Extract Untreated by Resin Adsorption 2000 L of water is added to 100 kg of persimmon leaves cut into strips, heated to 90 to 95° C., solid-liquid separated, and the extract (1) is obtained. Obtained. 400 L of water was added to the residue after solid-liquid separation, the residue was washed, and solid-liquid separation was performed to obtain an extract (2). The extracts (1) and (2) were combined and filtered, and the obtained filtrate was dried under reduced pressure at 80° C. to obtain a persimmon leaf extract not treated with resin adsorption.

Production Example 2: Production of resin-adsorbed persimmon leaf extract The filtrate obtained by the same procedure as in Production Example 1 was treated with 100 L of an adsorbent (HP-20) containing a styrene-divinylbenzene copolymer resin. The liquid was passed through the packed column. After washing with 400 L of water, the adsorbate was desorbed using 350 L of 80 (vol/vol)% ethanol. The desorbed liquid was filtered through a 0.45 μm filter and concentrated under reduced pressure at 45 to 50° C. to obtain a concentrated liquid. The concentrate was dried under reduced pressure at 80° C. to obtain a resin-adsorbed persimmon leaf extract. The yield was about 3-4%.

Test Example 1: Composition analysis of persimmon leaf extract not treated with resin adsorption and persimmon leaf extract after resin adsorption treatment Test Example 1-1: Method for measuring polyphenol content Polyphenols in persimmon leaf extract When the class content ((+)-catechin conversion) was measured three times (n = 3) by the Folin-Ciocalteu method, it was 46.7% by mass, 48.4% by mass, and 45.7% by mass.

Test Example 1-2: Method for measuring water content When the water content of the persimmon leaf extract was measured by the loss-on-drying method (1 g, 105°C, 6 hours), it was 7% or less by mass.

Test Example 1-3: Method for Measuring Contents of Fructose, Glucose and Sucrose The contents of fructose, glucose and sucrose in the persimmon leaf extract were measured three times (n=3) by HPLC. The HPLC measurement conditions were as follows.

[HPLC operating conditions]
Detector: RI
Column: CAPCELL PAK NH2 UG80 S5
Mobile phase: acetonitrile/water mixture (80:20)
Flow rate: 1.0 mL/min

The results are shown below.
First time:
Glucose 0.11g/100g, fructose 0.05g/100g or less, sucrose 0.2g/100g or less Second time:
Glucose 0.13g/100g, fructose 0.05g/100g or less, sucrose 0.2g/100g or less 3rd time:
Glucose 0.08g/100g, fructose 0.05g/100g or less, sucrose 0.2g/100g or less

Test Example 2: Measurement of glucose uptake promoting activity using skeletal myotube cells C2C12 mouse skeletal myoblasts stored frozen were thawed and transferred to a 96-well clear-bottom black microtiter plate (manufactured by Falcon). was seeded at a concentration of 5000 cells/200 μl/well and cultured in high glucose DMEM medium (5790) to obtain differentiated myotube cells. After replacing the medium with low-glucose DMEM medium (6047) and performing starvation, the persimmon leaf extract not treated with resin adsorption or the persimmon leaf extract after resin adsorption treatment obtained in Production Example 1 or 2. The medium was replaced with low-glucose DMEM medium containing 100 μg/ml or 200 μg/ml of . A low-glucose DMEM medium to which nothing was added was used as a negative control. After 60 minutes of incubation, medium was removed and 100 μM photolabeled non-hydrolyzable sugar analog, 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6- The medium was replaced with a low-glucose DMEM medium in which deoxyglucose (6-NBDG; manufactured by Invitrogen) was dissolved, and reacted for 30 minutes. After the reaction, the medium was discarded, and after washing with PBS twice, the well-drained microplate was measured at 444 nm/538 nm emission using a fluorescence plate reader. The measurement results are shown in FIG.

As is clear from FIG. 1, it was found that the persimmon leaf extract after resin adsorption treatment has the effect of improving the sugar metabolism function in skeletal muscle compared to the persimmon leaf extract not treated with resin adsorption. .

Claims (5)

Polyphenols obtained by adsorbing a water extract of persimmon leaves to DIAION (registered trademark) HP-20 and eluting with a water-ethanol mixture containing 50 to 80 (vol / vol)% ethanol. 20% by mass or more based on the total amount of the composition, and a water content of 10% by mass or less based on the total amount of the composition. 2. The persimmon leaf extract according to claim 1, wherein the total content of fructose, glucose and sucrose is 1.5% by mass or less relative to the total amount of the composition. A persimmon leaf water extract is adsorbed on DIAION (registered trademark) HP-20 and eluted with a water-ethanol mixture containing 50 to 80 (vol / vol)% ethanol. A method for producing an extract. A food, medicine, or cosmetic using the persimmon leaf extract according to claim 1 or 2. A composition for improving sugar metabolism, comprising the persimmon leaf extract according to claim 1 or 2.

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