JP6131164B2 - PPAR activator and composition thereof - Google Patents

Description

  The present invention relates to PPAR activators and compositions thereof. Specifically, it is related with the composition containing the extract extracted from the miso produced | generated in the miso manufacturing process, and an extract.

  PPAR (Peroxisome Proliferator Activated Receptor, peroxisome proliferator activated receptor) is a protein (nuclear receptor) that exists in the nucleus of a cell. There are three subtypes of PPAR, PPARα, PPARβ, and PPARγ, and it is known that PPARα and PPARγ contribute to activation of lipid metabolism in the body and improvement of insulin resistance in the metabolic syndrome. In addition, a component that activates PPAR that controls sugar / lipid metabolism present in foods is expected to be effective in preventing and improving metabolic syndrome.

  Patent Document 1 discloses a PPAR activator containing a solanaceous plant extract. Patent Document 2 discloses a PPAR activator containing a late glycation product (AGE) obtained from an organic solvent extract of miso.

JP 2011-184411 A JP 2012-87093 A

  As described above, it is known that food extracts have PPAR activity effective in improving lifestyle-related diseases. As a result of earnestly examining whether or not a substance having further excellent PPAR activity can be obtained, the inventor has conceived that an extract extracted from miso produced in the miso manufacturing process has excellent PPAR activity.

  That is, an object of the present invention is to provide a PPAR activator containing an extract extracted from miso and a composition thereof.

  In order to achieve the above object, the PPAR activator of the present invention has the following constitution. That is, the present invention is a PPAR activator comprising an extract extracted from miso, wherein the extract contains one or more substances selected from glycosides, fatty acid ethyl esters, and isoflavones. Features. According to this configuration, an excellent PPAR activator can be obtained by the glycoside, fatty acid ethyl ester and isoflavone contained in the extract extracted from miso.

  Moreover, in this invention, it is preferable that the color of the said miso is 1-23 in the Y value of XYZ color system. According to this, the PPAR activator contained in miso increases and an excellent PPAR activator is obtained.

  Moreover, in this invention, it is preferable that the said miso is a rice miso or a bean miso. According to this, an excellent PPAR activator can be obtained by increasing the number of PPAR activators from miso prepared from other raw materials.

  Moreover, in this invention, it is preferable that the said extract contains a browning substance further. According to this, compared with the extract which does not contain a browning substance, a PPAR activator increases further and the PPAR activator which was excellent is obtained.

  In addition, the composition according to the present invention is characterized by containing any of the PPAR activators described above. According to this, the PPAR activator effective for reducing neutral fat and suppressing obesity can be applied to various forms such as seasonings, food additives, functional foods and supplements.

  ADVANTAGE OF THE INVENTION According to this invention, the PPAR activator containing the extract extracted from miso and its composition can be provided.

It is a graph which shows the change of the blood glucose level of the mouse | mouth which ingested the feed using the extract of the miso which concerns on embodiment of this invention. It is a graph which shows the blood glucose level increase amount from the 0th day to the 35th day of the blood glucose level on the 35th day of the mouse | mouth which ingested the feed using the extract of the miso which concerns on embodiment of this invention. It is a graph which shows the change of the neutral fat value in the mouse | mouth plasma which ingested the feed using the extract of the miso which concerns on embodiment of this invention. It is a graph which shows the increase amount of the triglyceride value from the 35th day of the neutral fat value of the 35th day of the mouse | mouth which ingested the feed using the extract of the miso which concerns on embodiment of this invention, and the 0th day to the 35th day. . It is a graph which shows the neutral fat value in the liver of the 35th day of the mouse | mouth which ingested the feed using the extract of the miso which concerns on embodiment of this invention. It is a fraction data of an extract obtained by extracting 5-year-aged rice miso with 70% ethanol.

Hereinafter, embodiments of the present invention will be described in detail.
The PPAR activator according to this embodiment contains an extract extracted from miso. Various types of miso have been made using soybeans as the main ingredient, changing the amount of grains such as rice and wheat, salt, and koji. The extract of miso extracted from these miso contains PPAR activator. include. The PPAR activator includes a PPARα activator having an effect of reducing neutral fat and a PPARγ activation inhibitor having an effect of suppressing obesity.

  The PPAR activating substance contained in the miso extract of this embodiment contains one or more compounds, and contains one or more substances selected from glycosides, fatty acid ethyl esters, and isoflavones. Furthermore, browning substances may also be included. Examples of the sugar moiety constituting the glycoside include glucose and fructose. Examples of the fatty acid ethyl ester include linolenic acid ethyl ester. Examples of isoflavones include daidzein and genistein.

  In addition, the miso used in the present embodiment is not particularly limited in the ratio of soybeans and koji, and the amount of salt is not particularly limited, and any miso produced according to a normal method for producing miso. Or a commercial product thereof. And when manufacturing miso, the aging period when making it age | cure after preparation tends to contain many PPAR activation substances, so that it is long. Further, the color of miso containing a large amount of PPAR activator is 1 to 23 in the XYZ color system, and the value tends to decrease as the ripening period increases, and the color of miso becomes darker and reddish. Increase. The extract of miso extracted from this miso contains a large amount of PPAR activator and exhibits higher PPAR activity. The miso is preferably a rice miso made from soybean and rice as a raw material cereal or a bean miso made only from soybean. More preferably, the miso has increased redness, and the miso has a Y value in the range of 1 to 6.

  The extract of miso used in this embodiment may be extracted from the miso using a polar solvent. In addition to water, examples of the polar solvent include alcohols such as ethanol, and extraction can be performed using only water, a mixed solution of water and ethanol, or ethanol alone.

  The composition containing the PPAR activator of this embodiment can be applied to foods and pharmaceuticals such as seasonings and supplements, and can be ingested or administered to obtain a PPAR activity effect. When a composition containing a PPAR activator is used as a seasoning, miso is also included, and a miso extract may be added to the miso. In addition, it is possible to ingest a PPAR activator by ingesting miso, and for example, it can be easily ingested by eating it as miso soup.

  Further, in the extract of miso contained in the PPAR activator of the present embodiment, a glycoside having a PPAR activation ability, a fatty acid ethyl ester, and an isoflavone may each be present alone in the extract of miso, These substances may be combined and contained comprehensively. These substances are useful in addition to the effects on metabolic syndrome, and are more effective for maintaining health.

  Hereinafter, although an example is given and explained, this embodiment is not limited to these.

  Table 1 shows the miso used in the present embodiment. The rice miso of Nos. 1 to 6 differs in the type of miso because the blending ratio of the raw materials including the dry weight ratio of rice and soybean is different, and further differs in the color (Y value) of miso because the aging period is different.

[Method for producing rice miso of No. 1-6]
An appropriate amount of 5 to 10 kg of rice, salt and water was used as a raw material so that the weight ratio of Table 1 was 10 kg per soybean. To the cooked soybeans, rice cooked with koji fungi (rice koji) was added, further mixed with sodium chloride, fermented and aged to produce No. 1-6 miso.

[Method for producing bean miso of No. 7]
As a raw material, 10 kg of soybeans, an appropriate amount of salt and water were used, and salt was mixed with soybeans that had been boiled and cultured with koji mold (bean koji), which was fermented and aged to produce a bean miso.

[Method for producing No. 8 wheat miso]
As raw materials, 10kg soybean, 12kg barley, salt and water were used in an appropriate amount, and the soybeans cooked with barley or bare wheat and cultivated koji mold (wheat straw), and further mixed with salt, Was fermented and matured to produce a barley miso.

[Method for producing No. 9 white miso]
10kg soybean, 25kg rice, salt and water used as raw materials Heated before and after and decomposed and aged for several days.

[Measurement method of miso color]
The color of the miso was measured using a colorimetric color difference meter (manufactured by Nippon Denshoku Industries Co., Ltd.) (C light source). The Y value of the XYZ color system (Yxy color system) was measured, and the measurement results are shown in Table 1.

  For the extract of miso, 1 g of miso was placed in a tube with a lid, 10 mL of 70% ethanol was added, and the mixture was shaken and stirred for 10 minutes using a forced shaker. After stirring, centrifugation (12000 rpm) was carried out, and the supernatant was used as an extract of miso (hereinafter sometimes referred to as an extract).

[PPAR ligand assay]
experimental method:
Recombinant CV1 cells obtained by transforming CV1 cells with PPARα expression plasmid 1.0 μg, P4 × UAS Sg-tk-luc (luciferase expression plasmid) 5.0 μg, PRL-CMV (internal standard) 0.05 μg were added to miso. The extract was added and cultured in a CO ₂ incubator at 30 ° C. for 24 hours. After culturing for 24 hours, a cell disruption buffer (Passive Lysis buffer) was added, and the cells were lysed while shaking the plate at 20 ° C. for 15 minutes. Cell lysate was added to the chemiluminescence plate at 10 μL / well, and luciferase activity was measured using a luminometer (Bertreude).

  Table 2 shows the results of the PPARα ligand assay values contained in the miso concentration of 100 mg / L using the extraction solvent as the miso concentration of 100 mg / L. Change the extraction solvent (100% ethanol: extraction of hydrophobic substances, 70% ethanol: extraction of hydrophobic and hydrophilic substances, water: extraction of hydrophilic substances) to make miso extracts, and for each extract A PPARα ligand assay was performed.

  In any miso and each extraction solvent, the PPARα-activating substance was obtained, and in particular, the amount of the PPARα-active substance increased in the extract extracted from the rice miso of No. 6 that was aged for 5 years and the bean miso of No. 7. Moreover, the amount of the extract extracted with ethanol from No. 4 rice miso and the amount of the extract extracted with water from No. 8 wheat miso increased.

[Reduction effect of neutral fat using FAO cultured cells]
experimental method:
FAO cells were replaced with a medium containing 0.5 mM oleic acid and 20 μg / mL BSA, and incubation was performed for 24 hours. The medium was replaced with a PPAR ligand-added medium, and after incubation for 24 hours, the culture supernatant was removed and the cells were washed with PBS. Neutral fat was extracted with hexane-isopropanol (3: 2), and triglyceride E-Test Wako (Wako Pure Chemical Industries) was used to analyze the amount of neutral fat. Further, the cells were collected, the protein in the cells was measured by the Bradford method, and the amount of neutral fat per cell protein was calculated.

  As a result of testing the effect of reducing triglyceride using rat liver FAO cells, triglyceride was reduced, and especially for rice miso of No. 6 aged for 5 years and No. 7 of bean miso, the amount of neutral fat was reduced. Increased. As described above, it was found that the miso extract was ripened for a long period of time and the extract of miso obtained had a higher neutral fat reducing effect by increasing the color of the miso and increasing the redness.

[PPARγ activation inhibition]
About each miso extract, competitive inhibition with respect to troglidazone which is an agonist of PPARγ was examined at a miso concentration of 100 mg / L, and PPARγ activation inhibition was examined. As a result, it was confirmed that the water extract of 5-year-aged miso of No. 6 inhibits PPARγ activation.

[Confirmation of neutral fat reduction effect by animal test]
4-week-old kkAy mice (CLEA Japan) were fed with a high-fat diet supplemented with miso extract, and the effect of reducing neutral fat expected from PPAR ligand assay and cell test results was confirmed. As the miso used in the test, there are rice miso of No. 6 aged 5 years and red rice miso of No. 3, which have high PPARα activity and confirmed the effect of reducing neutral fat in cells.

  15 g of miso was suspended in 150 mL of distilled water and stirred for 15 minutes. The supernatant was obtained after centrifugation (11000 rpm, 10 minutes). An additional 150 mL of 70% ethanol was added to the residue and stirred for 15 minutes. Centrifugation was similarly performed, the supernatant was obtained, combined with the water extraction supernatant, and concentrated using a rotary evaporator. The concentrate was added to a mouse high fat diet (Research Diet D12492) so as to correspond to 2 and 6 miso soups to prepare a diet. Table 3 shows the bait composition. Since Table 3 is a rounded value, the total value is not necessarily 100.

  The daily consumption of miso soup was assumed to be two cups in the morning and evening. In this case, the daily consumption of miso is 32 g. As for the human body weight, the average body weight of 67.6 kg for Japanese males in their 30s and 60s was adopted because BMI in their 30s and 60s was markedly increased. The human body weight and the daily consumption of miso were extrapolated to mice, and the daily miso consumption of mice was calculated to be 9.5 mg. Since the amount of food per mouse per day is 3.42 g, the ratio of 9.5 mg miso added to 3.42 g of food is 0.28% high for low-dose food (equivalent to 2 cups of miso soup). In the case of a volume bait (equivalent to 6 cups of miso soup), the content was 0.84%. Table 4 shows the types of food. Each group received 8 test feeds for a test period of 35 days. Feeding was carried out by pair feeding.

[Eating and drinking]
Table 5 shows the average daily intake of feed. There was no significant difference between the test groups. In addition, Table 6 shows the average daily amount of drinking water. Drinking was free drinking. Compared with the non-administration subject group (C), the 5-year-aged miso-administered group (AL, AH) showed a tendency that the amount of drinking water decreased depending on the dose of miso. In contrast, the red rice miso-administered group (BL, BH) showed no significant change in the amount of drinking water compared to the non-administered control group.

[Weight change]
As a result of measuring the change in body weight during the test period, there was a tendency that the 5-year-aged miso-administered group and the red rice miso-administered group showed a decrease in body weight and weight gain compared to the non-administered control group.

[Changes in blood glucose level]
FIG. 1 shows the change in blood glucose level during the test period. (A) of FIG. 1 is a 5-year-aged miso administration group, and (b) is a red rice miso administration group. FIG. 2 shows the blood glucose level on the 35th day and the increase in blood glucose level from the 0th day to the 35th day. In the 5-year-aged miso-administered group and the red rice miso-administered group, the blood glucose level and the increase in blood glucose level tended to decrease compared to the non-administered control group. In particular, a significant decrease in blood glucose level and increase in blood glucose level was observed in the 5-year-aged miso-administered group compared to the non-administered control group. FIG. 3 shows the change in the neutral fat level in plasma during the test period. FIG. 4 shows the triglyceride value on day 35 and the amount of triglyceride increase from day 0 to day 35. The increased amounts of neutral fat and neutral fat decreased in the 5-year-aged miso-administered group and the red rice miso-administered group compared to the non-administered control group.

[Liver changes]
The increase in liver weight is less in the 5-year-aged miso-administered group and the red rice miso-administered group than in the non-administered control group. FIG. 5 shows the value of neutral fat in the liver on the 35th day. The neutral fat in the liver was reduced in the 5-year-aged miso-administered group and the red rice miso-administered group compared to the non-administered control group.

[Identification of compounds]
After fractionating the miso extract with HPLC (liquid chromatography), each fraction was subjected to PPAR ligand assay. Further, LCMS (fractionation by liquid chromatography followed by substance identification by mass spectrometry) analysis was performed to identify the compound.

(1) 5-year-aged rice miso 70% ethanol extract Fig. 6 shows fraction data of an extract obtained by extracting 5-year-aged rice miso of number 7 with 70% ethanol. FIG. 6A shows the result of HPLC (chromatography) analysis, and FIG. 6B shows the result of ligand assay. PPARα activity was confirmed in the range of F1, F4, F9 and F10. F9 (mass number 255.1) and F10 (mass number 271.1) were daidzein and genistein peaks. Further, F4 is a substance having a mass number of 391.3, and LCMS analysis was performed on this peak. The compound at this peak was a glycoside.

A component analysis was conducted on a 5-year-aged miso 70% ethanol extract using GCMS (gas chromatography). As a result, it was found that C ₂₀ H ₃₆ O ₂ was present, which was a fatty acid ethyl ester. Since this compound could not be confirmed in the soybean 70% ethanol extract, it is considered to be produced in the miso fermentation process. Further, this fatty acid ethyl ester is contained in F9 and F10 having PPARα ligand activity in LCMS analysis.

  Furthermore, in order to analyze the structure of fatty acid ethyl ester, the extract of miso was investigated with a nuclear magnetic resonance apparatus (NMR). As a result, it was found that the fatty acid ethyl ester contained in the miso extract was a mixture of α-linolenic acid ethyl ester and γ-linolenic acid ethyl ester.

  From the above, the PPARα ligand activity was measured for the HPLC fraction of the miso extract, but in the miso extract, one compound was not highly active, but a plurality of compounds were active. LCMS analysis was performed on each fraction of the miso extract to identify daidzein and genistein. In combination with GCMS analysis, fatty acid ethyl ester (mixture of α-linolenic acid ethyl ester and γ-linolenic acid ethyl ester) was confirmed in the active fraction in the 70% ethanol extract fraction of 5-year-aged miso.

Claims (5)

A PPAR activator comprising an extract extracted from miso;
The PPAR activator, wherein the extract contains γ-linolenic acid ethyl ester as an active ingredient .   2. The PPAR activator according to claim 1, wherein the color of the miso is 1 to 23 in a Y value of an XYZ color system.   The PPAR activator according to claim 1 or 2, wherein the miso is rice miso or bean miso.   The PPAR activator according to any one of claims 1 to 3, wherein the extract further contains a browning substance.   A composition for activating PPAR, comprising the PPAR activator according to any one of claims 1 to 4.