JP5844529B2 - Blood GIP level increase inhibitor - Google Patents

Description

  The present invention relates to a blood GIP concentration increase inhibitor.

GIP (Gastric inhibitory polypeptide or glucose-dependent insulinotrophic polypeptide) is one of the gastrointestinal hormones and is secreted from K cells present in the small intestine when fed. GIP is known to have a gastric acid secretion inhibitory action and a gastric movement inhibitory action (see Non-Patent Documents 1 to 3).
In addition, GIP is known to promote insulin secretion from pancreatic β cells and enhance glucose uptake into adipocytes in the presence of insulin. Therefore, it is considered that the action of GIP is a factor in obesity. In fact, there is a report that obesity is suppressed when the function of GIP is inhibited (see Non-Patent Document 4).
Furthermore, it has been reported that GIP contributes to insulin resistance (see Non-Patent Document 4). When insulin resistance develops, the absorption of sugar by insulin decreases, resulting in hyperinsulinemia. Hyperinsulinemia is said to be the root cause leading to the development of various lifestyle-related diseases such as obesity, and prevention and improvement of insulin resistance is also from the aspect of reducing the risk of lifestyle-related diseases. is important.

Thus, if GIP can be effectively suppressed, effects such as digestion promotion, improvement of stomach upset, prevention / improvement of obesity and insulin resistance can be expected.
Based on previous studies, 3-bromo-5-methyl-2-phenylpyrazolo [1,5-a] pyrimidin-7-ol (BMPP) and pyrazolopyrimidine compounds are known as substances that inhibit the function of GIP. It has been. Moreover, guar gum etc. are known as what suppresses secretion of GIP after a meal (refer patent documents 1 and 2, nonpatent literatures 5-10). However, these substances are not sufficient in terms of safety and effectiveness.

  On the other hand, polyglutamic acid is widely used in the fields of food, medicine, cosmetics and the like as a moisturizing agent and absorbent because of its high water-holding power, and has attracted attention as a highly safe biodegradable polymer. In addition, it has been reported that polyglutamic acid has an effect of promoting calcium absorption from the small intestine and an effect of suppressing an increase in blood pressure (see, for example, Patent Documents 3 and 4). Moreover, in order to suppress an increase in blood sugar level, a blood sugar level improving agent using polyglutamic acid has been proposed (see Patent Document 5). Furthermore, it has been reported that polyglutamic acid has an effect of suppressing the absorption of neutral lipids and can be used for the treatment, improvement, and suppression of onset of hypertriglyceridemia (see Patent Document 6).

  However, there has been no report on the inhibitory action on the increase in blood GIP concentration related to a specific salt of polyglutamic acid.

International Publication No. 01/87341 Pamphlet JP 2006-213598 Gazette Japanese Patent Laid-Open No. 5-95767 JP 2008-255063 A JP 2005-200330 A JP 2009-173634 A

J. C. Brown et al., Canadian J Physiol Pharmacol 47: 113-114, 1969 J. M. Falko et al. Clin Endocrinol Metab 41 (2): 260-265, 1975 Toshiji Oda et al., Gastrointestinal function and pathology, 1981, Chugai Medical, P205-216 Miyawaki K et al., Nat Med. 8 (7): 738-42, 2002 Gagenby S J et al., Diabet Med. 13 (4): 358-64, 1996 Ellis PR et al., Br J Nutr. 74 (4): 539-56, 1995 Simoes Nunes C et al., Reprod Nutr Dev. 32 (1): 11-20, 1992 Morgan LM et al., Br J Nutr. 64 (1): 103-10, 1990 Requejo F et al., Diabet Med. 7 (6): 515-20, 1990 Morgan et al., Br J Nutr. 53 (3): 467-75, 1985

  An object of the present invention is to provide a blood GIP concentration increase inhibitor useful for pharmaceutical or food applications. Specifically, the present invention relates to reduction, prevention, improvement, alleviation, treatment, promotion of digestion, or development of obesity or insulin resistance caused by an increase in blood GIP concentration after meals. It is an object of the present invention to provide a blood GIP concentration increase inhibitor useful as a food or non-pharmaceutical use for risk reduction, prevention, improvement, mitigation, or treatment.

  The present inventors examined the GIP secretion inhibitory action of polyglutamic acid and its salts. The calcium salt of polyglutamic acid significantly suppressed postprandial GIP secretion, promoted digestion, improved stomach sag, or obesity and insulin resistance. It was found to be particularly useful for the prevention and improvement of sex. The present invention has been completed based on this finding.

  The present invention relates to a blood GIP concentration increase inhibitor containing a calcium salt of polyglutamic acid as an active ingredient.

  According to the blood GIP concentration increase inhibitor of the present invention, an increase in blood GIP concentration, particularly an increase in blood GIP concentration after a meal can be suppressed. Further, the blood GIP concentration increase inhibitor of the present invention effectively suppresses the increase in blood GIP concentration, thereby reducing, preventing, improving, mitigating, treating, promoting digestion, It is useful for reducing, preventing, improving, mitigating and treating the risk of developing obesity and insulin resistance.

Hereinafter, the present invention will be described in detail.
The blood GIP concentration increase inhibitor of the invention contains a calcium salt of polyglutamic acid as an active ingredient. Polyglutamic acid, in which the amino group of the carboxyl group and α-position of the γ-position of glutamic acid is a peptide bond, the formula is represented by (-NH-CH (COOH) -CH 2 -CH 2 -CO-) n However, the calcium salt of polyglutamic acid used in the present invention is one in which 50% or more of the hydrogen atoms of the carboxyl group in the structural formula are replaced with calcium. The calcium salt of polyglutamic acid used in the present invention is preferably one in which 60% of the hydrogen atoms of the carboxyl group in the structural formula are replaced by calcium, and more than 70% of the hydrogen atoms of the carboxyl group, It is preferable that 80% or more, further 90% or more, further 95% or more, and especially 99% or more are substituted by calcium, but substantially all carboxyl groups are substituted by calcium. Particularly preferred. Moreover, it is preferable that the carboxyl group which will be located at the terminal in the structural formula is also substituted with calcium. Moreover, it is preferable that the carboxyl group which will be located at the terminal in the structural formula is also substituted with calcium.
The calcium salt of polyglutamic acid has the effect of significantly suppressing an increase in blood GIP concentration compared to polyglutamic acid and other polyglutamic acid salts. Therefore, the calcium salt of polyglutamic acid can be used as a GIP concentration increase inhibitor in blood, and can be used to produce the GIP increase inhibitor. Until now, it has not been known that the calcium salt of polyglutamic acid has an action of suppressing an increase in blood GIP concentration. In addition, it is not known that calcium salt of polyglutamic acid has an effect of preventing or improving obesity or insulin resistance.

The blood GIP concentration increase inhibitor of the present invention can be suitably used particularly for suppressing an increase in blood GIP concentration after a meal. Specifically, it is preferably used for suppressing an increase in blood GIP concentration after ingesting a diet containing both lipid and carbohydrate, particularly a diet rich in lipids, especially a diet rich in triacylglycerol. Can do.
The triacylglycerol contained in the meal is not particularly limited, and specific examples of the lipid component rich in triacylglycerol include butter, lard, fish oil, corn oil, rapeseed oil, olive oil, sesame oil and the like.
There are no particular restrictions on the carbohydrate components contained in the meal, and specific examples include cooked rice, starch, wheat flour, sugar, fructose, glucose, glycogen and the like.
In addition, the intake amount of the lipid and carbohydrate is not particularly limited as long as it is within the range included in a normal meal.
In the present invention, “inhibition of increase in blood GIP concentration” mainly means suppression of increase in blood GIP concentration that occurs after meals. The suppression of the increase in blood GIP concentration after a meal does not necessarily mean that the increase in blood GIP concentration caused after a meal is completely suppressed, but the blood GIP concentration increase inhibitor of the present invention is administered. It is a concept that also includes a moderation of the degree of increase in blood GIP concentration compared to the case where there is no blood GIP. And the “blood GIP concentration increase inhibitory action” in the present invention is a GIP secretion inhibitory action that suppresses the increase in blood GIP concentration by suppressing GIP secretion from the gastrointestinal tract, and a decrease in blood GIP concentration. It is a concept that includes any GIP lowering action that suppresses an increase in blood GIP concentration.

The calcium salt of polyglutamic acid can generally have an inhibitory effect on the increase in blood GIP concentration regardless of its molecular weight.
The calcium salt of polyglutamic acid used in the present invention preferably has a weight average molecular weight of about 500 or more, more preferably 1,000 or more, still more preferably 2,000 or more. More preferably, it is more preferably 6,000 or more, and particularly preferably 8,000 or more.
The upper limit of the weight average molecular weight of the calcium salt of polyglutamic acid to be used is preferably about 5,000,000. However, when the preparation for inhibiting the increase in blood insulin concentration of the present invention is used as an oral liquid preparation From the standpoints of throat scrubbing, sliminess, ease of swallowing, etc., it is preferable that the viscosity is relatively low, and the weight average molecular weight of the calcium salt of polyglutamic acid used is 1,000,000 or less Is more preferably 500,000 or less, further preferably 100,000 or less, particularly preferably 50,000 or less, and particularly preferably 30,000 or less.
More specifically, the weight average molecular weight of the calcium salt of polyglutamic acid used in the present invention is preferably 500 to 5,000,000, more preferably 1,000 to 1,000,000. Is more preferably 4,000 to 100,000, still more preferably 4,000 to 100,000, particularly preferably 6,000 to 50,000, further 8,000 to 30,000, It is especially preferable that it is 8,000-20,000.
The weight average molecular weight can be measured, for example, by high performance liquid chromatography using a gel filtration column.

The calcium salt of polyglutamic acid used in the present invention is a polyglutamic acid or salt thereof produced by chemical synthesis or microorganisms, or a commercially available polyglutamic acid or salt thereof, as described in the examples below. It can be obtained by neutralizing with an aqueous calcium oxide solution. It can also be produced by microorganisms cultured in a mixed medium containing calcium. The optical activity of glutamic acid constituting the calcium salt of polyglutamic acid may be D-form, L-form, or a mixture thereof. Polyglutamic acid naturally glutamic acid is a polymer bound at position gamma, as the microorganism capable of producing poly glutamic acid in wild-type, for example, a portion of Bacillus (Bacillus) bacteria including Bacillus natto and its allied species ( Bacillus subtilis var. Chungkookjang , Bacillus licheniformis , Bacillus megaterium , Bacillus anthracis , Bacillus halodurans ), Natrialba aegyptiaca , Hydra, etc. [Ashiuchi, M., et al .: Appl. Microbiol. Biotechnol., 59, pp.9-14 (2002)]. Also, genetically as the production example of the polyglutamic acid with the engineering technique, about 9 g / L / 5 days in transgenic recombinant Bacillus subtilis at plasmid (Bacillus subtilis ISW1214 strain) [Ashiuchi, M., et al .: Biosci. Biotechnol. Biochem., 70, pp. 1794-1797 (2006)], about 4 g / L / 1.5 days in recombinant Escherichia coli transfected with a plasmid [Jiang, H., et al .: Biotechnol. Lett., 28, pp.1241-1246 (2006)] is known to be obtained. Alternatively, polyglutamic acid is commercially produced as a food additive, cosmetic material, thickener and the like, and polyglutamic acid supplied by domestic and overseas polyglutamic acid manufacturers can be purchased (for example, domestic manufacturers: Nippon Polyglu, Ichimaru Falcos, Meiji Food Materia, etc., overseas manufacturers: BioLeaders, etc.).

  The calcium salt of polyglutamic acid used in the present invention can suppress GIP secretion after ingesting carbohydrates, lipids and / or proteins, thereby suppressing an increase in blood GIP concentration. And the inhibitory effect is remarkably excellent compared with polyglutamic acid and other polyglutamate.

  The blood GIP concentration increase inhibitor of the present invention may be the calcium salt of polyglutamic acid itself. In addition, the blood GIP concentration increase inhibitor of the present invention is a suitable liquid or solid excipient or extender such as titanium oxide, calcium carbonate, distilled water, lactose, starch, etc., in addition to the calcium salt of polyglutamic acid. May be included. The content of the calcium salt of polyglutamic acid in the blood GIP concentration increase inhibitor of the present invention is not particularly limited, but is preferably 0.01 to 100% by mass, and preferably 0.1 to 95% by mass. More preferably, it is contained in an amount of 1 to 90% by mass, particularly preferably 10 to 85% by mass.

  When the blood GIP concentration increase inhibitor of the present invention is used for foods, pharmaceuticals, etc., the calcium salt of polyglutamic acid alone is administered to humans and animals in the digestive tract, intraperitoneal, intravascular, intradermal, In addition to administration by subcutaneous administration or the like, it can be ingested as an active ingredient in various foods, pharmaceuticals, pet foods and the like. The concept of food is general food, as well as reduction, prevention, improvement, alleviation and treatment of stomach upset risk, promotion of digestion, and reduction, prevention, improvement, alleviation and treatment of obesity and insulin resistance. If necessary, it can be applied to foods such as beauty foods, foods for the sick, and foods for specified health use. When used as a pharmaceutical, for example, oral solid preparations such as tablets and granules, and oral liquid preparations such as oral liquids and syrups can be used.

  When preparing an oral solid preparation, an excipient, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, a corrigent and the like were added to the calcium salt of polyglutamic acid. Thereafter, tablets, coated tablets, granules, powders, capsules and the like can be produced by conventional methods. Moreover, when preparing a liquid preparation for oral use, a liquid preparation, a syrup, an elixir, etc. can be manufactured by a conventional method by adding a corrigent, a buffer, a stabilizer, a corrigent and the like.

The blending amount of the calcium salt of polyglutamic acid in each food or preparation is not particularly limited, but is preferably 0.01 to 100% by mass, more preferably 0.03 to 90% by mass, and More preferably, it is contained in an amount of 1-80% by mass, particularly preferably 0.3-70% by mass, and particularly preferably 1-60% by mass.
The effective administration (intake) amount in each food or preparation is preferably 0.001 g / kg body weight to 1.0 g / kg body weight per day as a calcium salt of polyglutamic acid, 0.003 g / kg body weight to The weight is more preferably 0.5 g / kg body weight, and still more preferably 0.01 g / kg body weight to 0.2 g / kg body weight. Moreover, the blood GIP concentration increase inhibitor of the present invention can be used before, during or after a meal, but is preferably used before or during a meal, and more preferably from 1 hour before the meal.
The subject of administration or ingestion is not particularly limited as long as it is a person who needs it, but fasting blood glucose level is 100 mg / dL or more, fasting blood triglyceride value is 100 mg / dL or more, and / or fasting. Those whose blood GIP value is 10 pg / mL or more are preferred. In addition, obese and metabolic syndrome patients and their preparatory groups are also preferable as administration or ingestion subjects. As a standard for obesity, in Japan, the standard BMI is set to 22. Therefore, a person with BMI = 22 or more is preferable, and a person with BMI = 25 or more is more preferable. On the other hand, in Europe and the United States, as a standard of obesity, BMI of 25 or more is overweight, so those with BMI = 25 or more are preferable, and those with BMI = 30 or more are more preferable. According to the diagnostic criteria of metabolic syndrome, in the case of Japanese, the waist is 85 cm or more for men and 90 cm or more for women, and (1) blood triglyceride is 150 mg / dL or more or HDL cholesterol is 40 mg / A person who has one or more of the following three items: less than dL, (2) hyperglycemia (fasting blood glucose is 110 mg / dL or more), and (3) hypertension (130/85 mHg or more) Since those who are in the reserve group and who apply to two or more items are considered to fall under the metabolic syndrome, it is preferable that these persons be subjects for administration or ingestion. In the case of the United States, metabolic syndrome is considered to satisfy at least three of abdominal circumference (102 cm or more for men, 88 cm or more for women), high neutral fat, low HDL, high blood pressure, and high fasting blood glucose. From these, it is preferable that these persons, including those in the reserve group corresponding to two or more, be subjects for administration or ingestion.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

[Analysis method]
Quantitative determination and weight average molecular weight determination of polyglutamic acid calcium salt:
The weight average molecular weight of the calcium salt of polyglutamic acid was measured by gel filtration using a D-6000 (manufactured by Hitachi High-Technologies Corporation) HPLC system. The analysis conditions were as follows: TSKGel G3000PWXL gel filtration column (trade name, manufactured by Tosoh Corporation) was used as the analysis column, 0.1 M sodium sulfate was used as the eluent, the flow rate was 0.8 mL / min, the column temperature was 50 ° C., and UV detection was performed. The wavelength was 210 nm. The weight average molecular weight was determined using polyglutamic acid (Meiji Food Materia Co., molecular weight 9k), polyhydroxyproline (SIGMA-) whose weight average molecular weight was previously determined using pullulan (trade name: Shodex STANDARD P-82, manufactured by Showa Denko KK). Measurement was performed using ALDRICH, molecular weight 4k) as a standard product.

[Preparation Example] Preparation of polyglutamic acid calcium salt Using a sodium salt of polyglutamic acid having a weight average molecular weight of 9,000 (manufactured by Meiji Food Materia Co., Ltd.) as a starting material, 125 mL of a 20% (w / w) aqueous solution was prepared and cooled on ice. Was adjusted to pH 2 or lower using hydrochloric acid. Subsequently, the generated acid precipitate was recovered by centrifugation at 8,000 rpm for 5 minutes (trade name: himacCR21GIII, manufactured by Hitachi Koki Co., Ltd.), and the resulting precipitate was washed with the same amount of distilled water. And again subjected to centrifugation. After repeating this washing operation twice, the obtained precipitate was suspended in 300 mL of distilled water, and neutralized with calcium hydroxide so that the pH was 7 or more. This acid treatment and neutralization treatment with calcium hydroxide were performed again, 2.5 times the amount of ethanol was added to the obtained neutralized sample, and the mixture was allowed to stand overnight under ice cooling. The precipitate produced by the addition of ethanol was collected by centrifugation (at the same as above) at 14,000 rpm for 5 minutes, and the collected sample was dried under reduced pressure to obtain 19.0 g of a solid as a calcium salt. The weight average molecular weight of this sample was calculated to be 11,000 by the above analysis method.

[Test Example] Polyglutamic acid calcium salt suppresses blood triglyceride concentration from rising: Polyglutamic acid calcium salt having a weight average molecular weight of 11,000 (prepared in Preparation Example 1) and sodium salt of polyglutamic acid having a weight average molecular weight of 9,000 (Meiji Food Materia Co., Ltd.) was used to prepare a sample for oral administration as follows.
In addition, an oral administration test was carried out as follows using seven 8-week-old male mice (C57BL / 6J Jcl: manufactured by Clea Japan) in each group.

Preparation of orally administered samples:
Glucose (manufactured by Kanto Chemical Co., Inc.) and triolein (Glyceryl trioleate: manufactured by Sigma) are emulsified using lecithin (manufactured by egg, manufactured by Wako Pure Chemical Industries, Ltd.) and albumin (derived from bovine serum, manufactured by Sigma) to prepare an emulsion. did. A polyglutamate sample was added to this emulsion, and the final concentrations were 5 (w / w)% polyglutamate sample, 5 (w / w) glucose, 5 (w / w) triolein, emulsifier (lecithin 0). .2 (w / w)%, albumin 1.0 (w / w)%), orally administered samples were prepared. In addition, the sample which added water instead of the polyglutamate as a control sample was prepared.

Oral administration test:
Mice fasted overnight were subjected to initial blood collection using an heparinized hematocrit capillary tube (VITREX) from the orbital vein under ether anesthesia. Thereafter, the orally administered sample was orally administered with an oral sonde needle, and blood was collected from the orbital vein under ether anesthesia after 10 minutes, 30 minutes, 1 hour and 2 hours. The oral dose for mice is shown in Table 1 below.

Blood collected with a heparinized hematocrit capillary was stored under ice cooling until plasma separation, and then centrifuged at 11000 rpm for 5 minutes to obtain plasma. From the obtained plasma, the blood GIP concentration was measured using a Rat / Mouse GIP (Total) ELISA kit (manufactured by Linco Research / Millipore co., ELISA method).
As a result of measuring the blood GIP concentration up to 2 hours after oral administration of the sample, it was found that the blood GIP concentration reached the maximum 10 or 30 minutes after administration. Therefore, the difference (Δ value) between the maximum value of blood GIP concentration (10 minutes or 30 minutes after administration) and the initial value (at the time of initial blood collection) is defined as the increase in maximum GIP concentration, and the value when the control group is 100 Are shown in Table 2.

  Based on the obtained maximum GIP concentration increase value, a statistically significant difference between groups was also examined, and the results are also shown in Table 2. When significance (P <0.05) was recognized by analysis of variance, multiple control test (Bonferroni / Dunn method) was performed between the control group and each polyglutamate administration group, and the sodium salt administration group and calcium salt. The test was conducted with the administration group, and the significance was judged from the obtained results with P <0.05 as a significant difference.

  From the results in Table 2, it was found that the polyglutamic acid calcium salt significantly suppressed the increase in blood GIP concentration compared to the control group. Furthermore, the effect was remarkably excellent as compared with the sodium salt of polyglutamic acid.

  As described above, GIP is known to play a role in inhibiting gastric acid secretion and gastric motility, increasing glucose uptake into adipocytes in the presence of insulin, and inducing insulin resistance. Therefore, the calcium salt of polyglutamic acid can be suitably used for the promotion of digestion, prevention and improvement of stomach sag, prevention and improvement of obesity and insulin resistance by effectively suppressing an increase in blood GIP concentration. it can.

Claims (2)

  A blood GIP concentration increase inhibitor comprising a calcium salt of polyglutamic acid as an active ingredient.   A blood GIP concentration increase inhibitor comprising a calcium salt of polyglutamic acid.