JP6977945B2 - A composition for promoting GLP-1 secretion and a method for producing the composition. - Google Patents

Description

The present invention relates to a GLP-1 secretagogue composition used for promoting glucagon-like peptide-1 (GLP-1), which is a kind of incretin, from the intestine, and a method for producing the composition.

GLP-1 is a hormone endocrine from L-cells present in the epithelium of the intestine, and has an action of promoting insulin secretion associated with sugar intake and suppressing an increase in postprandial blood glucose level. In addition, GLP-1 has an action of promoting the proliferation of pancreatic β cells that control insulin secretion and an action of protecting the proliferation, and various actions on the liver, adipocytes, cardiovascular system, and nervous system have been reported. .. Although the secreted GLP-1 has the above-mentioned activity, it is known that it is converted to the inactive form within a short time by the action of dipeptidyl peptidase IV (DPP-IV) present in blood and tissues. ing. Therefore, the search for a GLP-1 receptor agonist that is not affected by DPP-IV and the study of inhibiting DPP-IV to sustain the action of GLP-1 are attracting attention in the field of pharmaceuticals for type 2 diabetes. In the food field, studies on dietary components that increase the secretion of GLP-1 with ingestion are underway.
Glutamine, which is an amino acid, and whey protein are known as components that promote the secretion of GLP-1.
Further, it has been found by the studies of the present inventors that a hydrolyzate of zein, which is a corn protein, a hydrolyzate of rice endosperm protein, and a hydrolyzate of rice bran protein strongly promote the secretion of GLP-1 (for example, Patent Document). 1 and Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2011-182742

Ishikawa et al. , Food Funct 2015; 6: 2525.

However, in order for the GLP-1 secretion promoting function of the dietary component to be exhibited, it is required to ingest a large amount of the dietary component. In addition, for that reason, ingesting a large amount of sugar and lipid is not suitable for the purpose of improving sugar and lipid metabolism. Therefore, a component having high GLP-1 secretion promoting activity is required.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a GLP-1 secretion-promoting composition having excellent GLP-1 secretion-promoting activity, and a method for producing the composition.

As a result of diligent studies, the present inventors have found that globulin, which is a protein fraction soluble in saline, has a strong GLP-1 secretion-promoting activity among rice germ milk proteins, and that the pepsin digested product of rice germ milk globulin is present. GLP-1 concentration in the portal blood of animals treated with globulin is significantly higher than that of animals not treated with the pepsin digested product of rice germ milk globulin and animals treated with the pepsin digested product of non-globulin protein. Furthermore, in a sugar loading test of an animal to which a globulin pepsin digested product was administered, it was found that the increase in blood glucose level of an animal to which sugar was intraperitoneally administered was significantly suppressed, and the present invention was completed.

Specifically, the present invention is as follows.
The first aspect of the present invention is a composition for promoting GLP-1 secretion containing rice endosperm globulin as an active ingredient.
A second aspect of the present invention is a composition for promoting GLP-1 secretion in which the content of rice endosperm globulin with respect to the amount of rice endosperm protein is 15% by mass or more.
A third aspect of the present invention is a composition for promoting GLP-1 secretion containing an alkali-extracted rice endosperm protein of a rice variety having a rice endosperm globulin content of 1.5 times or more as compared with ordinary domestic rice. Is.
A fourth aspect of the present invention is a method for producing a composition for promoting GLP-1 secretion, which comprises a step of adding rice endosperm globulin to rice endosperm protein.
A fifth aspect of the present invention is for promoting GLP-1 secretion, which comprises a step of obtaining an alkali-extracted rice endosperm protein from a rice variety having a rice endosperm globulin content of 1.5 times or more as compared with ordinary domestic rice. It is a method for producing a composition.

The GLP-1 secretagogue composition of the present invention is excellent in GLP-1 secretagogue activity.
According to the present invention, ingestion of rice germ milk globulin increases the amount of GLP-1 secretion, and as a result, the increase in postprandial blood glucose level can be suppressed, which contributes to the prevention and treatment of diabetes caused by the decrease in GLP-1 secretion. Can provide foods or medicines to be used.

It is a figure which shows the result of having compared the amount of GLP-1 secretion by GLUTag cells for each sample. It is a figure which shows the GLP-1 concentration in portal vein blood 15 minutes after administration of various samples. It is a figure which shows the graph which compared the blood glucose level after administration of various samples in the intraperitoneal glucose tolerance test (IPGTT).

Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments.

<Composition for promoting GLP-1 secretion and method for producing the composition>
The GLP-1 secretion-promoting composition according to the first aspect of the present invention contains rice endosperm globulin as an active ingredient.
The composition for promoting GLP-1 secretion according to the second aspect of the present invention has a content of rice endosperm globulin in an amount of 15% by mass or more based on the amount of rice endosperm protein.
The GLP-1 secretion-promoting composition according to the third aspect of the present invention comprises an alkali-extracted rice endosperm protein of a rice variety having a rice endosperm globulin content of 1.5 times or more as compared with ordinary domestic rice. contains.
The method for producing a GLP-1 secretagogue composition according to a fourth aspect of the present invention includes a step of adding rice endosperm globulin to rice endosperm protein.
The method for producing a GLP-1 secretagogue composition according to a fifth aspect of the present invention is an alkali-extracted rice from a rice variety having a rice endosperm globulin content of 1.5 times or more as compared with ordinary domestic rice. Includes the step of obtaining endosperm protein.

(Rice endosperm protein)
In the present invention, "rice endosperm protein" refers to rice protein extracted from rice endosperm.
Rice germ milk protein is obtained by treating polished rice or rice flour with an alkali to neutralize the protein solution extracted to recover the precipitate, or by allowing the polished rice or rice flour to act on amylases to hydrolyze the starch. It can be prepared by a method of recovering the remaining components.
(Rice endosperm globulin)
In the present invention, "rice endosperm globulin" refers to globulin extracted from polished rice, rice flour or rice endosperm protein with saline.
In the present invention, the "rice germ globulin" may be a digested product in which rice germ globulin is partially decomposed by a proteolytic enzyme such as a digestive enzyme (for example, pepsin).
The main component of rice endosperm globulin is α-globulin with a molecular weight of 26,000 daltons. Rice germ globulin is extracted by immersing rice germ milk or its pulverized product in 0.5 to 5 M saline solution. Under this condition, albumin extracted with pure water is also extracted at the same time, so the raw material is previously used. It is preferable to extract by immersing in a 0.5 to 5 M saline solution using a raw material extracted with water and from which albumin has been removed.

Polished rice or rice flour is extracted with water, and the extract is removed by centrifugation or filtration. Then, 0.5 to 5 M saline solution is added to the residue to extract, and the extract is recovered by centrifugation or filtration. Even when rice endosperm protein is used as a raw material, it is preferable to perform saline extraction after water extraction in the same manner.
Examples of the method for recovering the extracted globulin from the extract include a method of drying after dialysis using a dialysis membrane, a method of desalting and concentrating using an ultrafiltration membrane, and the like, and globulin in saline solution. Since globulin has low solubility under acidic conditions, it is possible to recover the refined product of rice germ milk globulin by adding an acid to the globulin extract to precipitate globulin and then recovering it by centrifugation or filtration.
It is preferable that at least a part of the rice endosperm globulin in the present invention is a refined product of the rice endosperm globulin obtained as described above.

The content of rice germ globulin with respect to the amount of rice germ protein is usually around 10% by mass, but in order to promote the secretion of GLP-1 with a lower intake of rice germ protein, rice germ milk as the composition of the present invention. The content of rice embryo milk globulin with respect to the amount of protein is preferably 15% by mass or more, more preferably 20% by mass or more, further preferably 50% by mass or more, and preferably 80% by mass or more. Especially preferable.
The upper limit of the content of rice endosperm globulin is not particularly limited, but is preferably 98% by mass or less.
The method for achieving the above-mentioned content of rice endosperm globulin with respect to the amount of rice endosperm protein is not particularly limited, and examples thereof include adding rice endosperm globulin to rice endosperm protein.
The content of rice germ globulin relative to the amount of rice endosperm protein shall be calculated by subjecting the obtained refined rice germ globulin to SDS-PAGE and measuring the content of α-globulin of 26 k Dalton with a densitometer. Can be done.

Among the rice varieties, low glutelin rice such as Chunyang, LGC1, and LGC soft is known. Most of these low glutelin rices have a rice endosperm globulin content of 1.5 times or more (for example, about 1.5 to 2 times) as compared with ordinary domestic rice (Koshihikari, Sasanishiki, etc.). Since rice endosperm protein extracted from such varieties of rice can promote GLP-1 secretion with a smaller intake than normal rice endosperm protein intake, rice using saline solution is used. The composition recovered by the usual method for extracting rice endosperm protein can also be used as it is without performing the fractional extraction of the endosperm globulin.

A composition containing a peptide obtained by degrading rice embryo milk globulin as it is or by using a proteolytic enzyme (for example, a digestive enzyme such as pepsin) can be used as a food or pharmaceutical for promoting GLP-1 secretion.
The form of the GLP-1 secretagogue composition of the present invention is not particularly limited as long as it contains rice germ milk globulin as an active ingredient, and is a rice cracker composition, powder (powder), liquid (for example, beverage), supplement. Foods such as, pharmaceuticals, etc. may be mentioned, and rice itself may be used.
For example, when the form of the composition for promoting GLP-1 secretion of the present invention is rice, the secretion of GLP-1 can be promoted by ingesting rice as a normal staple food.
Here, the rice cracker composition is a composition that composes at least a part of rice crackers produced from rice grains or rice flour as a raw material. Examples of the rice cracker composition include rice crackers, rice crackers, hail, okaki, kakimochi, rice snacks, and cereals made from rice or rice flour.

When the form of the GLP-1 secretagogue composition of the present invention is a pharmaceutical product, it can be orally administered. The administration method can be appropriately selected according to the patient's age and symptoms. The dose varies depending on the age, route of administration, and frequency of administration, and can be appropriately selected by those skilled in the art.
The dose when orally administered is not particularly limited, but is generally in the range of about 0.1 μg to 10 mg per 1 kg of body weight at a time.

Since the above-mentioned foods or pharmaceuticals can suppress an increase in postprandial blood glucose level, they can contribute to the prevention and treatment of diabetes caused by a decrease in GLP-1 secretion.
In particular, the composition of the present invention exerts an immediate effect of suppressing an increase in blood glucose level within 30 minutes (preferably within 15 minutes) at the latest after administration based on GLP-1 secretion. Therefore, it may be administered immediately after a meal, but after a lapse of 15 minutes or more after a meal (preferably after a lapse of 30 minutes or more, more preferably after an lapse of 1 hour or more), the blood glucose level has sufficiently risen. However, it is expected to have an immediate effect of suppressing the rise in blood glucose level. In addition, since it is derived from rice, it is expected to be a food or pharmaceutical product that suppresses an increase in blood glucose level due to a decrease in GLP-1 secretion that does not stress the patient.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Preparation of rice endosperm protein]
50 kg of rice flour derived from domestically produced polished rice (product name "Powder Rice DK", manufactured by Niigata Seifun Co., Ltd.) was suspended in 200 L of 0.2% sodium hydroxide solution and left at 20 ° C. overnight. The obtained suspension was supplied to a centrifuge (product name "H-130I", manufactured by Kokusan Co., Ltd.) at a flow rate of about 5 L / min and centrifuged at a rotation speed of 1,400 rpm. The outflowing centrifugal supernatant was collected and centrifuged again under the same conditions to obtain a protein extract from which starch granules had been removed. 6N hydrochloric acid was added to this extract to adjust the pH to 5.5. The protein aggregates were recovered by centrifugation under the above conditions, suspended in 100 L of water, and then the same centrifugation operation was performed again to obtain wet protein. This was suspended in 30 L of water, sterilized by heating at 80 ° C. for 30 minutes, homogenized, and spray-dried to obtain rice protein powder (protein purity 90%). The rice protein powder corresponds to rice endosperm protein.

[Protein fraction]
50 kg of powder rice DK was suspended in 200 L of tap water and left at 20 ° C. overnight. The obtained suspension was centrifuged under the same conditions as above. The outflowing centrifugal supernatant was collected and centrifuged again under the same conditions to collect the supernatant and use it as an albumin extract. This liquid was concentrated with an ultrafiltration membrane (AHP3010 manufactured by Asahi Kasei Corporation) having a molecular weight cut off of 50,000, ammonium sulfate was added so as to be 50% saturated, and then the precipitate was recovered. The precipitate was recovered by centrifugation, suspended in distilled water, desalted using an ultrafiltration membrane (AHP1010 manufactured by Asahi Kasei Corporation), and freeze-dried to obtain an albumin fraction.

The albumin extract was suspended in a 0.2% sodium hydroxide solution in the precipitate obtained when it was separated, and left at 20 ° C. overnight. The obtained suspension was treated with the same centrifuge as described above to collect the supernatant, which was then centrifuged again under the same conditions to obtain a protein extract from which starch granules had been removed. 3.2 kg of salt was added and dissolved in 160 L of this solution, and 6N hydrochloric acid was further added to adjust the pH to 10.0. Centrifuge under the same conditions as above to collect the precipitate (residual fraction), and the supernatant is further transferred to a Kokusan cooling high-speed centrifuge H-2000B equipped with a continuous rotor QNS at a flow rate of about 2 L / min. The precipitate was removed by continuous centrifugation at a rotation speed of 15,000 rpm. The pH of the centrifugation supernatant was adjusted to 3.0, and the precipitated precipitate was recovered by the same continuous centrifugation. The precipitate was suspended in 5 L of distilled water, stirred at room temperature for 1 hour, and then the precipitate was recovered by centrifugation again and freeze-dried to obtain a globulin fraction.
The residue fraction was suspended again in 100 L of water and then centrifuged under the same conditions to recover the precipitate, which was then freeze-dried to obtain a glutelin + prolamin fraction.

[Preparation of rice peptide]
2 g each of rice endosperm protein, albumin fraction, globulin fraction, and glutelin + prolamin fraction were suspended in 100 mlL of distilled water, and phosphoric acid was added to adjust the pH to 1.85. To this was added 10 mg of pepsin (derived from pig stomach, manufactured by Sigma) and reacted at 37 ° C. for 30 minutes. Calcium hydroxide was added to the reaction solution to neutralize the pH to 7.0. The enzyme was inactivated by treatment in boiling water for 10 minutes, and the supernatant was recovered by centrifugation. The collected supernatant was frozen overnight, thawed, centrifuged again to remove the precipitate, and lyophilized to recover the peptide.
In the present specification, peptides obtained from the rice endosperm protein, albumin fraction, globulin fraction, and glutelin + prolamin fraction are referred to as REPH, AlbuminH (AlbH), GlobulinH (GlbH), and Glutelin + ProlaminH (GluProH), respectively.

[Cell test]
Mouse colon-derived GLUTag cells (provided by Dr. Drucker, University of Toronto) were cultured in 48-well plates for 2 days until subconfluent, and Hepes buffers (140 mM NaCl, 4.5 mM KCl, 20 mM Hepes, 1.2 mM CaCl ₂ , 1, Wells were washed with .2 mM MgCl ₂ , 10 mM D-glucose, 0.1% BSA, pH 7.4).

80 μL of each solution of the sample (REPH, AlbaminH, GlobulinH, Glutellin + ProlaminH) dissolved in the same buffer at a concentration of 5 mg / mL was added, and the mixture was incubated at 37 ° C. for 60 minutes.
Peptide-free buffer was similarly added and incubated as a negative control (Sample Blk in FIG. 1).
After collecting the supernatant, centrifuge (800 × g, 4 ° C for 5 minutes), collect 60 μL of the supernatant, and set the GLP-1 concentration in the supernatant to a commercially available ELISA kit (EZGLP1T-36K, manufactured by Millipore). ).
FIG. 1 is a diagram showing the results of comparing the amount of GLP-1 secreted by GLUTag cells for each sample.
As is clear from the results shown in FIG. 1, significantly higher GLP-1 secretion was observed in all the samples as compared with the negative control sample Blk, but when compared with the addition amount of 5 mg / mL. It can be seen that Globulin H secreted a particularly high GLP-1 compared to others.

[Measurement of GLP-1 in portal blood]
9-week-old male SD rats were acclimatized for 1 week and subjected to the test at 10-week-old. The animals were fasted overnight from the day before the test, and a solution of 0.5 g of rice protein and 0.5 g of pepsin digested product thereof in 10 mL of distilled water was orally administered at 10 mL per kg of rat body weight. After 15 minutes, the abdomen was opened under anesthesia, portal vein blood was collected, and the GLP-1 concentration was measured. The results of statistical analysis of the test results for 6 to 7 animals in each group are shown in FIG.
As is clear from the results shown in FIG. 2, a significantly higher concentration of GLP-1 was observed only in the Globulin H-administered group.

[Intraperitoneal glucose tolerance test (IPGTT)]
7-week-old male SD rats were acclimatized for 1 week and subjected to the test at 8 weeks of age. The animals were fasted overnight from the previous day, and 10 mL of a solution of rice protein and 0.5 g of pepsin digested product of each fraction in 10 mL of distilled water was orally administered by gavage (the number of animals in each group was 6 to 6). 9 animals). Immediately after, glucose was intraperitoneally administered at a dose of 1 g / kg body weight. After 15, 30, 60, 90 and 120 minutes, blood was collected from the tail vein and the glucose concentration was measured.
As is clear from the results shown in FIG. 3, blood glucose levels were significantly lower in all sample administration groups 30 minutes and 60 minutes after administration than in the control group, and significantly lower in the Glucagon H administration group even 15 minutes after administration. Since the blood glucose level was shown, it was considered that the GLP-1 secretory activity contributed to the rapid effect of Globulin H on the blood glucose level.

Claims (12)

A GLP-1 secretagogue composition comprising only rice endosperm globulin as an active ingredient. The composition according to claim 1, wherein at least a part of the rice endosperm globulin is a refined rice endosperm globulin product. A fast-acting GLP-1 secretion-promoting composition having a content of rice endosperm globulin in an amount of 15% by mass or more based on the amount of rice endosperm protein and which can be effective within 15 minutes after administration. The fast-acting GLP-1 secretion-promoting composition according to claim 3, which contains an alkali-extracted rice endosperm protein of a rice variety having a content of rice endosperm globulin 1.5 times or more that of domestic rice Koshihikari. thing. Claimed that the above-mentioned "rice variety having a rice endosperm globulin content of 1.5 times or more as compared with domestic rice Koshihikari" is at least one selected from the group consisting of Chunyang, LGC1 and LGC soft. 4. The composition for promoting GLP-1 secretion according to 4. The composition according to any one of claims 1 to 5, which is a quick-acting type for administration within 60 minutes after a meal. The composition according to any one of claims 1 to 6, which is a food for promoting GLP-1 secretion. The composition according to any one of claims 1 to 6, which is a pharmaceutical product. A fast-acting GLP-1 secretion-promoting composition that can be effective within 15 minutes after administration, which comprises a step of adding rice endosperm globulin to rice endosperm protein so as to be 15% by mass or more with respect to the amount of rice endosperm protein. Production method. The production method according to claim 9, further comprising a step of obtaining an alkali-extracted rice endosperm protein from a rice variety having a content of rice endosperm globulin 1.5 times or more as compared with domestic rice Koshihikari. Claimed that the above-mentioned "rice variety having a rice endosperm globulin content of 1.5 times or more as compared with domestic rice Koshihikari" is at least one selected from the group consisting of Chunyang, LGC1 and LGC soft. 10. The method according to 10. The method according to any one of claims 9 to 11, wherein the composition is a composition for administration within 60 minutes after a meal.

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