JP2018102202A - Green leaf powder and composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oral green leaf powder having myoblast activation effect, muscle cell proliferation promotion effect and adipocyte differentiation inhibition effect and excellent in diet effect such as muscle enhancement and prevention or reduction of obesity, and a composition using the same.SOLUTION: The green leaf powder is a powder consisting of green leaf and having silicon content of 1,000 ppm or more by mass basis. There is also provided a beverage composition for green soup containing the green leaf powder with silicon content of 1,000 ppm or more by mass basis. There is also provided a composition for diet containing the green leaf powder with silicon content of 1,000 ppm or more by mass basis.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a green leaf powder, a composition using the same, and a method for producing the green leaf powder.

  Conventionally, compositions containing green leaf powder are known as health foods such as green juice. As raw materials such as green juice, various plants such as barley, tomorrow, and kale are known (Patent Document 1).

JP 2004-298199 A

With regard to green leaf powder used in health foods, etc., the needs of consumers have diversified in recent years, and there is an increasing demand for further improvement in effect, particularly in terms of health and beauty. ing.
However, the conventional green leaf powder and the composition containing the same have not sufficiently met this requirement.

  Then, this inventor earnestly examined about the structure which can obtain the further effect | action reinforcement | strengthening from a viewpoint of health or beauty about green leaf powder and a composition containing the same. As a result, surprisingly, the green leaf powder containing a certain amount or more of silicon content and the composition containing the same have a myoblast activation effect, a muscle cell differentiation promoting effect, and an adipocyte differentiation inhibiting effect, It has been found that it can be useful for activation and enhancement, suppression of adipose tissue increase, metabolism promotion and obesity prevention and reduction, thereby completing the present invention.

  The present invention is based on the above findings and provides an oral green leaf powder having a silicon content of 1,000 ppm or more on a mass basis.

  The present invention also provides a food and drink composition for green juice containing green leaf powder, wherein the silicon content of the powder is 1,000 ppm or more on a mass basis. is there.

  The present invention also provides a diet composition containing green leaf powder, wherein the silicon content of the powder is 1,000 ppm or more on a mass basis.

  The present invention also provides a cosmetic composition containing green leaf powder, wherein the powder has a silicon content of 1,000 ppm or more on a mass basis.

  According to the present invention, a green leaf powder having a myoblast activation effect, a muscle cell differentiation promoting effect and an adipocyte differentiation inhibiting effect, and having excellent diet effects such as muscle enhancement, metabolism promotion, obesity prevention or reduction, and the like. The used food and drink composition for green juice is provided. Moreover, according to this invention, the composition for diets excellent in the myoblast activation effect, the muscle cell differentiation promotion effect, and the adipocyte differentiation suppression effect can be provided.

FIG. 1 is a graph showing the myoblast activation activity test results of Examples and Comparative Examples. FIG. 2 is a graph showing Myogenin gene expression test results in myoblasts according to Examples and Comparative Examples. FIG. 3 is a graph showing the results of a Col1A1 gene expression test after induction of adipocyte differentiation of 3T3-L1 cells according to Examples and Comparative Examples.

  Hereinafter, the green leaf powder and composition of the present invention and the method for producing the green leaf powder will be described based on preferred embodiments thereof. The green leaf powder of this embodiment is an oral green leaf powder having a silicon content of 1,000 ppm or more on a mass basis. Moreover, the composition of this embodiment contains the green leaf powder whose silicon content is 1,000 ppm or more on a mass basis. Hereinafter, the composition of the present embodiment applies to any of the composition for eating and drinking for green juice, the composition for diet and the cosmetic composition.

  Green leaves are leaves of green plants. The green leaf of this embodiment itself is green. As a green plant, what is suitable for food-drinks use and can give a specific high silicon content to a green leaf powder is selected. From this point of view, in the present embodiment, particularly preferable examples of the green plant include grasses such as wheat, rice, sweet potato, persimmon, fin, acne, corn, sorghum and sugar cane. As the gramineous plant, wheat is preferable because of its particularly high effect of promoting the differentiation of muscle cells. As the barley, barley, wheat, oats, rye and kumazasa are preferably mentioned, and barley is most preferred from the viewpoint of dieting action. As barley, Nijo barley, Rojo barley, bare barley and the like can be used without particular limitation.

  In the present embodiment, the green leaf may include not only a leaf portion of a plant body but also a stem together with the leaf. Therefore, the green leaf powder may contain stem powder. In this case, if it is shown that the silicon content of the green leaf powder including the stem powder is 1,000 ppm or more on the mass basis, the silicon content of the green leaf powder is assumed to be 1,000 ppm or more on the mass basis. The same applies to the preferable silicon content range described later. In addition, the stalk here may contain the side bud.

  As described above, the green leaf powder in the present embodiment contains 1,000 ppm or more of silicon on a mass basis. Thereby, the green leaf powder has high myoblast activation effect, muscle cell differentiation promoting effect and adipocyte differentiation inhibiting effect. In addition, the silicon content of the green leaf powder is 1,000,000 ppm or less on the basis of mass, so that the myoblast activation effect and the muscle cell differentiation promoting effect, particularly the myoblast activation effect, and the fiber amount of the green leaf powder It is preferable because it is easy to be taken orally by reducing the amount. From these viewpoints, more preferably, the green leaf powder contains 3,000 ppm or more and 100,000 ppm or less silicon on a mass basis, and more preferably contains 5,000 ppm or more and 80,000 ppm or less silicon on a mass basis, Particularly preferably, 7,000 ppm or more and 50,000 ppm or less of silicon is contained on a mass basis. The silicon content of the green leaf powder can be measured by the method described in Examples described later. In order to make the silicon content of the green leaf powder within the above range, for example, the green leaf powder may be produced by a suitable method for producing the green leaf powder described below. In particular, in the process, green leaf powder having the above-mentioned silicon content, particularly green leaf powder containing more than 7,000 ppm of silicon on a mass basis can be easily produced by producing a powdered or pulverized powder having a high fiber content. It becomes easy to obtain.

  In the green leaf powder of the present embodiment, silicon is usually present in the form of silicon dioxide (silica).

  The green leaf powder preferably has a water content of 20% by mass or less, particularly 10% by mass or less from the viewpoint of stability and prevention of quality deterioration. For example, the water content is preferably 1% by mass or more from the viewpoint of ease of production of the green leaf powder.

  The green leaf powder is preferably a powder that passes through any of 30 to 250 mesh sieves in terms of ease of mixing with other components and ease of oral administration. From the same viewpoint, it is more preferable that 90% by mass or more of the green leaf powder passes through 200 mesh.

  As green leaf powder, various processed products of green leaf can be used. Examples of such processed products include dry pulverized powder obtained by subjecting green leaves to drying treatment and pulverization treatment (sometimes referred to simply as “dry powder”), and pulverization treatment obtained by subjecting green leaves to fragmentation treatment. Examples include powders, squeezed powders obtained by drying green leaf juice, and extract powders obtained by drying green leaf extract. From the viewpoint of making it easier to obtain a silicon content within the above range, green leaves The dry pulverized powder and the powdered product of the green leaf are preferable, and the dry pulverized powder of green leaves is most preferable from the viewpoint of easy production and ease of oral administration. Naturally, green leaf powder includes granular and granular forms. The green leaf powder may be a granulated product.

  From the viewpoint of further enhancing the myoblast activation effect, the muscle cell proliferation promoting effect, and the adipocyte differentiation inhibiting effect, the content of the green leaf powder in the solid content of the composition of the present embodiment is 5% by mass in dry mass. The above is preferable, 10% by mass or more is more preferable, 15% by mass or more is further preferable, and 20% by mass or more is particularly preferable. In addition, the composition of the present embodiment may be composed only of green leaf powder, but from the viewpoint of achieving high functionality and multi-function in combination with other components, the green leaf powder of the composition As an upper limit, 90 mass% or less is preferable, 85 mass% or less is more preferable, and 80 mass% or less is still more preferable.

  The composition of this embodiment may contain other components in addition to the green leaf powder having a specific silicon content. Examples of the other components include vitamins, proteins, oligosaccharides, minerals, dairy products, processed plant products, microorganisms such as lactic acid bacteria, sugars, sweeteners, citric acid, acidulants, colorants, and brighteners. In addition, production agents such as talc, cellulose, calcium stearate and the like can be blended. Examples of other components include various excipients, binders, lubricants, stabilizers, diluents, extenders, emulsifiers, coloring agents, fragrances, food additives, seasonings and the like. Can do. The content of other components can be appropriately selected according to the form of the composition. In this embodiment, it is preferable that components other than the green leaf powder which has the specific silicon content contained in a composition are 90 mass% or less in solid content, and it is especially preferable that it is 80 mass% or less.

  The composition of the present embodiment may be in any form such as solid, semi-solid, and fluid. For example, solid forms include tablets, sticks, plates, blocks, solids, rounds, bowls, tablets, gummi, wafers, biscuits, cookies, cakes, chewables, sticks, etc. Is mentioned. Examples of the semi-solid form include a paste form, a jelly form, a cream form, and a gel form. Examples of the fluid form include a syrup form, a liquid form, a cream form, and a gel form.

  The food and drink composition for green juice of this embodiment will be described in detail below. The food and drink composition for green juice is a beverage containing green leaves as various processed products. Examples of the composition for eating and drinking for green juice include this beverage and a solid that is dispersed or dissolved in a liquid to obtain this beverage. In particular, when the composition is in the form of powder or granules and is ingested orally with a mixture mixed with water, it is suitable for long-term storage while preventing spoilage, and when the composition for eating and drinking is mixed with water. Is preferable because it is vivid. Further, when the composition is in a solid form, as described above, it is made into a liquid mixed with water, and can be taken orally, such as drinking the liquid. Accordingly, it may be taken orally as a solid. Further, not only water but also milk, soy milk, fruit juice drink, whey drink, soft drink, yogurt, hot cake mix and the like may be used. Moreover, it cannot be overemphasized that you may use as a supplement, health food, functional nutrition food, functional indication food, food for specified health, and a pharmaceutical. The food and drink composition for green juice includes smoothies and the like in addition to the generally known green juice products.

  The form of the composition for diet and the composition for beauty of this embodiment is not limited to the beverage mentioned as an example of the composition for eating and drinking for green juice and the solid form dispersed or dissolved in the liquid to obtain this beverage, Any form can be adopted. In addition, it goes without saying that the diet composition and cosmetic composition of the present embodiment may be used as supplements, health foods, nutritional functional foods, functional display foods, foods for specified health use, and pharmaceuticals.

Hereinafter, the suitable manufacturing method of the powder of this embodiment is further demonstrated.
In the production method of the present embodiment, a grass plant is cultivated in volcanic ash soil, and green leaves of the grass plant are pulverized.
Volcanic ash soil contains a lot of crystalline or amorphous silicic acid and has a high silicon supply capacity to plants. Gramineae plants are plants with high silicon absorption. For this reason, according to the manufacturing method of this embodiment, it becomes easy to obtain the green leaf powder containing said silicon content. It is known that volcanic ash soil generally contains 10% by mass or more of silicon, for example, about 20-50% by mass in terms of SiO ₂ .
As the volcanic ash soil, black mysterious soil, moist black mysterious soil and black mysterious soil are known, and black mysterious soil is particularly preferable from the viewpoint of the growth of gramineous plants. Kuroboku soil is black soil composed of volcanic ash soil and humus as the base materials. Since the surface layer has a lot of humus, the color is black or black brown, and the lower layer is brown. It is often seen on plateaus and flatlands at the foot of the volcano.

  The black soil is not particularly limited as long as it is a commonly known black soil, but for example, it is mainly composed of volcanic ash soil and humus and has a color close to black. It is known to be the soil. Moreover, compared with red soil, black meso soil has a small content of effective phosphoric acid, and N: P ratio is about 2-5: 12-17. Therefore, when a gramineous plant is cultivated with black soil, excessive phosphorus supply to the gramineous plant can be avoided. Furthermore, black soil has about twice as much cation exchange amount as red soil. As a result, black soil tends to contain an abundant amount of exchangeable lime, exchangeable clay, and exchangeable potassium compared to red soil.

  The production method of the present embodiment may cultivate gramineous plants using only volcanic ash soil, but may include some soil other than volcanic ash soil, such as red soil and yellow soil. When cultivating with mixed soil of volcanic ash soil and soil other than volcanic ash soil, the proportion of volcanic ash soil is preferably 50 parts by mass or more and 100 parts by mass or more with respect to 100 parts by mass of the mixed soil used. It is preferably 90 parts by mass or more.

  Cultivation is not particularly limited as long as barley is grown to the extent that leaves and stems can be harvested from seeds and seedlings by a method commonly known in the art, and conditions such as temperature and humidity other than the soil to be used Can be appropriately set by those skilled in the art.

  Since the green leaves of the gramineous plant to be collected affect the beauty of the composition, it is preferably dark green. Specifically, when a leaf color scale is used, the average value of the leaves of one gramineous plant is, for example, 4.0 or more, and preferably 4.5 or more.

  For example, in the case of wheat, green leaves are preferably harvested before the maturity period, that is, from the beginning of splitting to the beginning of heading. The green leaves are preferably treated immediately after harvesting. When time is required for processing, it is stored by storage means commonly used by those skilled in the art, such as cold storage, in order to prevent deterioration of green leaves.

  For example, conventionally known methods can be used to dry green leaves. As such a method, a method combining drying treatment and pulverization treatment with respect to green leaves can be used. Either the drying process or the pulverization process may be performed first, but the drying process is preferably performed first. Dry powdering may be combined with one or more treatments selected from treatments such as blanching treatment and sterilization treatment, if necessary. In addition, the number of times of pulverization may be one time or a combination of two or more processes, but it is preferable to combine a fine pulverization process that finely pulverizes after the coarse pulverization process.

  The blanching process is a process for keeping the green color of the green leaves vivid, and examples of the blanching process include a hot water process and a steaming process. In the blanching treatment, the green leaves are preferably treated in hot water or steam at 80 to 100 ° C., preferably 90 to 100 ° C. for 60 to 180 seconds, preferably 90 to 120 seconds. In addition, when performing hydrothermal treatment as a blanching treatment, the green color of green leaves can be made more vivid by dissolving carbonates such as magnesium carbonate and hydrogen carbonates such as sodium bicarbonate in hot water. This is preferable because it is possible. Moreover, as a steaming process, the intermittent steaming process which repeats the process which steams a green leaf with water vapor | steam, and the process which cools under normal pressure or pressurization is preferable. In the intermittent steaming process, the steaming process is preferably performed for 20 to 40 seconds, more preferably 30 seconds. The cooling treatment after the steaming treatment is preferably performed immediately, and the method is not particularly limited, but the method is not particularly limited, soaking in cold water, refrigeration, cooling with cold air, evaporative cooling with hot air, evaporative cooling combining hot air and cold air Etc. are used. Among these, evaporative cooling combining hot air and cold air is preferable. Such cooling treatment is performed so that the temperature of the green leaves is preferably 60 ° C. or lower, more preferably 50 ° C. or lower, and most preferably 40 ° C. or lower. Moreover, in order to produce a green leaf powder rich in nutritional components such as vitamins, minerals, and chlorophyll, it is preferable to repeat the intermittent steaming treatment 2 to 5 times.

  The sterilization treatment is usually a treatment for physically and chemically killing microbial cells using temperature, pressure, electromagnetic waves, chemicals and the like. When the branching process is performed in addition to the drying process and the pulverization process, the branching process is preferably performed before the drying process. Moreover, when performing a sterilization treatment in addition to the drying treatment and the pulverization treatment, the sterilization treatment is preferably performed after the drying treatment or before or after the pulverization treatment.

  The drying treatment is preferably a treatment for drying so that the water content of the green leaves is 10% by mass or less, particularly 7% by mass or less. This drying treatment can be performed by any method known to those skilled in the art, such as hot air drying, high pressure steam drying, electromagnetic wave drying, freeze drying, and the like. Drying by heating can be performed at a temperature and a time at which green leaves are not discolored by heating, preferably at 40 ° C to 140 ° C, more preferably 80 to 130 ° C.

  Examples of the pulverization treatment include a pulverization treatment by an arbitrary method commonly used by those skilled in the art using a crusher, a mill, a blender, a stone mortar or the like. The pulverized green leaves are sieved as necessary.

  As a specific dry powdering (pulverized powder) method, for example, after cutting green leaves, a blanching treatment is performed, and then dried so that the water content is 10% by mass or less, preferably 7% by mass or less. Then, a method of pulverizing is mentioned (see JP 2004000210 A). Further, for example, a method of cutting green leaves, performing branching treatment, then twisting, and then drying and pulverizing (see Japanese Patent Application Laid-Open No. 2002-0665204 and Japanese Patent No. 3428956) can also be mentioned. Further, for example, a method of drying green leaves, coarsely pulverizing them, heating them at 110 ° C. or higher, and further finely pulverizing them (see Japanese Patent Application Laid-Open No. 2003-033151 and Japanese Patent No. 3277181) can be mentioned.

  As a method for fragmenting the green leaves, methods commonly used by those skilled in the art to fragment a plant body, such as slicing, crushing, and chopping, can be used. As an example of fragmentation, a slurry may be used. Slurry is performed by putting barley green leaves through a mixer, a juicer, a blender, a mascoloider, etc., and making the barley green leaves into a cocoon (liquid and solid suspension). By slurrying in this way, the green leaves are preferably 80% by mass or more of the strips, preferably having an average diameter of 1 mm or less, more preferably 0.5 mm or less, even more preferably 0.1 mm or less, and most preferably 0.05 mm. It is made into pieces and has fluidity.

  Examples of the method of squeezing green leaves include a method of squeezing green leaves or a fragmented product thereof, or a method of centrifuging or filtering a fragmented product of barley green leaves. A typical example is a method of squeezing juice by a mechanical crushing means such as a mixer or a juicer, and obtaining a squeezed liquid by removing coarse solids by means such as sieving or filtration, if necessary. . Specific examples include the methods described in JP-A-08-245408, JP-A-09-047252, JP-A-5-7471, JP-A-4-341153, and the like. Can be appropriately selected and implemented by those skilled in the art.

  As a method for obtaining a green leaf extract, an extraction solvent usually used by those skilled in the art, such as ethanol, water, hydrous ethanol, or the like, is added to green leaf or a fragmented product thereof, and extraction is performed by stirring or heating as necessary. be able to. The extract may be concentrated as necessary.

The green leaf powder of the present embodiment has a specific silicon content, as described in the examples described later, and thereby has a myoblast activation effect, a myoblast differentiation promoting effect, and an adipocyte differentiation inhibiting effect. Specifically, by ingesting the green leaf powder of the present embodiment and a composition containing the same, it is possible to activate myoblasts in skeletal muscles and promote differentiation of myoblasts into muscle cells. Moreover, differentiation of adipocytes from fibroblasts can be suppressed. Therefore, since the composition of this embodiment can promote muscle strengthening and prevention of muscle reduction and promote metabolism by ingesting it, it can promote adipose tissue reduction and prevention of adipose tissue increase. In addition, obesity can be prevented or reduced.
Therefore, the composition of this embodiment is used for myoblast activation, muscle cell proliferation promotion, adipocyte differentiation suppression, muscle tissue activation, muscle cell proliferation promotion, muscle mass increase, adipose tissue reduction, metabolism. It can be excellent in promotion applications, obesity prevention applications, obesity prevention applications, diet applications, body fat reduction applications, and the like.
Moreover, the composition of this embodiment is useful also for the improvement of beauty by containing the green leaf powder containing the silicon more than the said specific amount.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further more concretely, the scope of the present invention is not limited to these Examples.

[Example 1]
Barley seeds were sown in a field of black soil (presumably containing about 30% by mass of silicon in terms of SiO ₂ ). Barley was cultivated by usual plant cultivation methods such as water supply and weed management. An example of soil analysis data of Kuroboku soil is shown in Table 1.
From the barley, green leaves including barley stems before heading were cut. This was washed with water to remove adhering mud and the like, and a pretreatment was performed to cut it to a size of about 5 to 10 cm. The pretreated green leaves were blanched only once with hot water at 90 to 100 ° C. for 90 to 120 seconds, and then cooled with cold water. Subsequently, the obtained green leaves were dried with warm air at 80 ° C. to 130 ° C. for 20 minutes to 180 minutes in a dryer until the water content became 10% by mass or less. The dried green leaves were roughly crushed to a size of about 1 mm. The obtained green leaves of barley were finely pulverized so that 90% by mass or more passed through the 200 mesh section to obtain a dry powder (ground powder) sample of barley foliage. The green leaf powder sample passed through 200 mesh was 90% by mass or more, and the water content was 1% by mass to 7% by mass.

[Example 2]
Various Kumazasa pulverized powders were screened based on the silicon content, and those having a silicon content of 34,900 ppm were used.

[Example 3]
Various barley green leaf pulverized powders were screened based on the silicon content, and those having a silicon content of 5,650 ppm were used.

[Comparative Example 1]
A commercially available powder of tomorrow leaves was used.

[Comparative Example 2]
Kale was cultivated in the same manner as in Example 1, and the leaf containing the obtained stem was treated in the same manner as in Example 1 to obtain a dry powder sample of green leaves. The green leaf powder sample that passed through 200 mesh was 90% by mass or more.

<Measurement of silicon content>
The silicon content of the obtained powder was measured using an ICP emission analysis method (measured at the Japan Food Analysis Center). The results are shown in Table 2.

The powders of Examples 1 to 3 and Comparative Example 1 were subjected to a myocyte activation test according to the following procedures (1) to (7).
[Myocyte activation test]
(1) Mouse skeletal muscle-derived myoblasts (product name C2C12, manufactured by RIKEN BioResource Center) are cultured in a 75 cm ² flask containing 10 vol% FBS-DMEM medium at 37 ° C in a 5% CO ₂ incubator. did.
(2) After culturing in (1), cells suspended by trypsin treatment were collected from a 75 cm ² flask, and after counting the number of cells, each well in a 96-well plate coated with collagen had a cell density of 4000 cells / well. After inoculating the whole medium, it was precultured for 24 hours in a 37 ° C., 5% CO ₂ incubator.
(3) Separately from (1) and (2), a solution obtained by dispersing or dissolving the powders of Examples 1 to 3 and Comparative Example 1 in a 10% by volume FBS-DMEM medium to a concentration of 500 μg / ml. A sample solution was prepared by sterilizing the filter using a 0.2 μm filter (manufactured by Advantech). As a control, 10 vol% FBS-DMEM medium itself was used as a sample solution.
(4) After removing the medium from each well, 200 μL each of the sample solution prepared in (3) was added to each well and cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours.
(5) After culturing in (4), the medium was removed, and each well was washed once with PBS 200 μL / well. Subsequently, 150 μL / well of Cell Counting Kit-8 solution (Dojin Chemical Co., Ltd.) diluted 30-fold with serum-free DMEM was added.
(6) The plate after the addition of the solution in (5) was allowed to stand in a 37 ° C., 5% CO ₂ incubator for appropriate color development, and then the absorbance of each well at 450 nm was measured. Based on the obtained data, the ratio of the number of cells to the control (% of control) was calculated based on the following formula, and this was defined as myoblast activation activity.
% of control = (Data sample-Data blank) / (Data control-Data blank) × 100
Data sample: Absorbance of Examples 1 to 3 and Comparative Example 1
Data control: Absorbance of control
Data blank: Blank when there is no cell

(7) Evaluation FIG. 1 shows a summary of the calculation results of the ratio of the number of cells in Examples 1 to 3 and Comparative Example 1 with the control as 100%.

  As shown in FIG. 1, according to the powder of each Example, the activation effect of the myocyte was acquired. Especially, the myocyte activation effect of barley green leaf powder was high. On the other hand, in each comparative example, the activation effect of myocytes was low.

  Next, the powders of Examples 1 to 3 and Comparative Examples 1 and 2 were subjected to the Myogenin gene expression test, which is a differentiation marker for myoblasts, according to the following procedures (a) to (f).

[Myogenin gene expression test]
(A) Culture of mouse skeletal muscle-derived myoblasts (product name C2C12, manufactured by RIKEN BioResource Center) at 37 ° C in a 5% CO ₂ incubator using a 75cm ² flask containing 10vol% FBS-DMEM medium. did.
(B) After culturing in (a), cells suspended by trypsin treatment were collected from a 75 cm ² flask and the number of cells was counted. Then, 2 × 10 ⁴ cells / well were added to each well in a collagen-coated 24-well plate. After inoculating the medium at the cell density, the cells were precultured for 96 hours in a 37 ° C., 5% CO ₂ incubator.
(C) Separately from (a) and (b), the powders of Examples 1 to 3 and Comparative Examples 1 and 2 were dispersed or dissolved in a 10 vol% FBS-DMEM medium to a concentration of 1000 μg / ml. A sample solution was prepared by sterilizing the solution using a 0.2 μm filter (Advantech). As a control, 10 vol% FBS-DMEM medium itself was used as a sample solution.
(D) After removing the medium from each well, 500 μL of the sample solution prepared in (c) was added to each well and cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours.
(E) After culturing in (d), each well was washed twice with 500 μL / well of PBS. Subsequently, mRNA was recovered using RNeasy Mini Kit 250 (manufactured by QIAGEN). Using the obtained mRNA as a template, cDNA was synthesized using ReverTra Ace (registered trademark) qPCR RT Master Mix with gDNA Remover (manufactured by Toyobo).
(F) Quantitative real-time PCR using QuantiNova SYBR Green PCR Kit (manufactured by QIAGEN) using the cDNA obtained in (e) as a template and the following primers (manufactured by QIAGEN) to determine the mRNA expression levels of Gapdh and Myogenin. It was measured.
Gapdh: Mm_Gapdh_3_SG QuantiTect Primer Assay (QT01658692)
Myog: Mm_Myog_1_SG QuantiTect Primer Assay (QT00112378)

  The analysis was performed by relative quantification, and the mRNA amount was corrected using the Gapdh mRNA expression level as an endogenous control. ) With respect to the corrected Myogenin gene expression level in each example and each comparative example, a relative value was calculated with the corrected gene expression (group in which differentiation induction was applied and no sample was added) as 1. The results are shown in FIG.

  As shown in FIG. 2, the Myogenin gene expression improving effect in myoblasts was obtained by the powder of each example. In particular, the improvement effect of barley green leaf powder was high. In contrast, in each of the comparative examples, myogenin gene expression in myoblasts was low.

Subsequently, the powders of Examples 1 to 3 and Comparative Examples 1 and 2 were subjected to an adipocyte differentiation inhibition test according to the following procedures (1) to (11).
[Adipocyte differentiation inhibition test by gene expression in 3T3-L1 cells]
(1) Using mouse fibroblast 3T3-L1 (RIKEN BioResource Center) in a 75 cm ² flask containing 10 vol% FBS-DMEM medium, in a 5% CO ₂ incubator until it reaches a predetermined number, 37 Culturing was carried out under humid conditions at ° C.
(2) The medium was removed from the flask in (1), washed 3 times with DPBS (manufactured by Nacalai Tesque), and the cells were detached by trypsin treatment.
(3) After stopping the trypsin reaction by adding fresh 10 vol% FBS-DMEM medium, the cells were collected in a tube and centrifuged at 800 rpm for 3 minutes to precipitate the cells.
(4) Suspend cells in fresh 10vol% FBS-DMEM medium to 2 × 10 ⁵ cells / mL, seed 100 μL each in a 96 well plate, and in a 5% CO ₂ incubator for 2 days, 37 Pre-cultured at a temperature of 0 ° C. under humid conditions.
(5) Apart from (1) to (4), IBMX (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in DMSO so that 0.5M and Dexamethasone (manufactured by Wako Pure Chemical Industries, Ltd.) were 1 mM. Test by adding 0.5 M IBMX and 1 mM Dexamethasone prepared above and 10 mg / mL insulin solution (manufactured by Sigma Aldrich) to 10 vol% FBS-DMEM medium to final concentrations of 0.5 mM, 1 μM and 10 μg / mL, respectively. A medium was prepared. A liquid in which the powders of Examples 1 to 3 and Comparative Examples 1 and 2 were dispersed or dissolved in the test medium so as to be 10 mg / ml was prepared, and this was filtered using a 0.2 μm filter (Advantech). The sterilized product was used as a sample solution. As a control, the test medium itself was used as a sample solution.
(6) Remove the medium from the 3T3-L1 cells pre-cultured in (4), add 100 μL of the sample solution prepared in (5) to each well, and in a 5% CO ₂ incubator for 1 day at 37 ° C. The cells were cultured under humid conditions.
(7) After 24 hours, the medium was removed from each well, and RNA was purified from the cells using Rneasy mini (Qiagen).
(8) ReverTra Ace (registered trademark) qPCR RT Master Mix with gDNA Rem
cDNA was synthesized by over (Toyobo).
(9) Rps28 (Mm_Rps28_1_SG QuantiTect Primer Assay, Qiagen) primer as internal standard, Col1A1 (Mm_Col1a1_1_SG QuantiTect primer assay, Qiagen) primer, QuantiNOVA as internal standard, using cDNA obtained in (8) as a template PCR was performed with Rotor-Gene Q (Qiagen) using SYBR GREEN (Qiagen).
(10) As a result of PCR, mRNA expression levels of Rps28 and Col1A1 were analyzed using Rotor Gene Q Pure Detection (manufactured by Qiagen). The analysis was performed by relative quantification, and the amount of mRNA was corrected using the amount of Rps28 mRNA expressed as an endogenous control.
(11) For the corrected Col1A1 gene expression level in each example and each comparative example, a relative value was calculated with the corrected gene expression (group in which differentiation was induced and no sample was added) as 1. . The results are shown in FIG.

As shown in FIG. 3, when fibroblast 3T3-L1 cells are induced to differentiate into adipocytes in the presence of the green leaf powder of each example, the expression of Col1A1 gene is improved in fibroblast 3T3-L1 after the induction treatment. The effect was obtained. In particular, the effect of improving the expression of barley green leaf powder was high. In contrast, in each of the comparative examples, the expression of the Col1A1 gene after induction of adipocyte differentiation was low. The Col1A1 gene is a fibroblast marker, and it is known that when the fibroblast 3T3-L1 is induced to differentiate into adipocytes, the expression level of the Col1A1 gene is drastically reduced very early. Therefore, a large Col1A1 gene indicates that differentiation into adipocytes is suppressed. From this test, it was shown that differentiation from fibroblasts into adipocytes is effectively suppressed by the green leaf powder of the present invention.

The present invention relates to green leaf powder and composition using the same.

Hereinafter, the green leaf powder and compositions and preferred method for producing the green leaf powder of the present invention will be described with reference to preferred embodiments thereof. The green leaf powder of this embodiment is an oral green leaf powder having a silicon content of 1,000 ppm or more on a mass basis. Moreover, the composition of this embodiment contains the green leaf powder whose silicon content is 1,000 ppm or more on a mass basis. Hereinafter, the composition of the present embodiment applies to any of the composition for eating and drinking for green juice, the composition for diet and the cosmetic composition.

Claims (5)

  A green leaf powder having a silicon content of 1,000 ppm or more on a mass basis.   The green leaf powder according to claim 1, wherein the green leaf is a green leaf of a gramineous plant.   A dietary composition for green juice containing green leaf powder having a silicon content of 1,000 ppm or more on a mass basis.   A composition for diet containing a green leaf powder having a silicon content of 1,000 ppm or more on a mass basis. A cosmetic composition comprising green leaf powder having a silicon content of 1,000 ppm or more on a mass basis.