JP2012144487A - Blood triglyceride concentration rise inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a blood triglyceride concentration rise inhibitor.SOLUTION: The blood triglyceride concentration rise inhibitor includes a calcium salt of polyglutamic acid as an active constituent.

Description

  The present invention relates to a blood triglyceride level increase inhibitor.

Triglycerides are a type of neutral lipid, and most of the neutral lipids contained in blood are triglycerides. In blood, it is known that hyperlipidemia is caused when a high triglyceride concentration continues. Hyperlipidemia is considered to be the cause of arteriosclerosis and is the first trigger to cause diseases such as heart disease and cerebrovascular disorder.
In general, changes in blood triglyceride concentration are strongly influenced by diet, and it is said that it is difficult to control completely with drugs alone. Therefore, the quality of lipids ingested as a diet more than drug therapy has attracted attention, and for example, it is recommended to reduce blood triglyceride levels by ingesting highly unsaturated fatty acids, mainly linoleic acid and linolenic acid. Yes. However, it has been pointed out that excessive consumption of polyunsaturated fatty acids leads to the production of peroxy fatty acids in the body and induces various adult diseases.
Under such circumstances, it is desired to suppress an increase in blood triglyceride concentration by a method that is highly safe and does not cause side effects even when administered or ingested daily. In recent years, as a substance that suppresses the increase in blood triglyceride concentration safely and effectively, chitansan gum, alginic acid propylene glycol ester (see Patent Document 1), chitosan (see Patent Document 2), and modified starch (see Patent Document 3) that suppress lipid absorption. ) Has been reported.

  On the other hand, polyglutamic acid is widely used as a moisturizing agent, an absorbent and the like because of its high water-retaining ability, and has attracted attention as a biodegradable polymer. In addition, it has been reported that there is an effect of promoting calcium absorption from the small intestine and an effect of suppressing blood pressure increase (see Patent Documents 4 and 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 so far on the action of suppressing the increase in blood triglyceride concentration related to a specific salt of polyglutamic acid.

JP-A-5-186356 JP-A-3-290170 JP 2004-269458 A Japanese Patent Laid-Open No. 5-95767 JP 2008-255063 A JP 2009-173634 A

  An object of the present invention is to provide a blood triglyceride level increase inhibitor useful for pharmaceutical or food applications. Specifically, the present invention suppresses an increase in blood triglyceride concentration, and suppresses an increase in blood triglyceride concentration that is useful as a pharmaceutical or food for preventing or improving hyperlipidemia or arteriosclerosis. It is an object to provide an agent.

  In view of the above problems, the present inventors have conducted intensive studies. As a result, the inventors have found that polyglutamic acid salts have an effect of suppressing an increase in blood triglyceride concentration after meals. Furthermore, it has been found that among the polyglutamic acid salts, the calcium salt has a particularly excellent effect of suppressing an increase in blood triglyceride. The present invention has been completed based on these findings.

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

  According to the blood triglyceride concentration increase inhibitor of the present invention, an increase in blood triglyceride concentration, particularly an increase in blood triglyceride concentration after a meal can be reduced. Furthermore, the blood triglyceride level increase inhibitor of the present invention adjusts the blood lipid level to a normal range, reduces, prevents, improves, alleviates and treats the risk of developing hyperlipidemia, and further prevents arteriosclerosis. Useful for reducing, preventing, improving, mitigating and treating the risk of onset.

Hereinafter, the present invention will be described in detail.
The blood triglyceride concentration increase inhibitor of the present 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.
The calcium salt of polyglutamic acid has an effect of significantly suppressing an increase in blood triglyceride concentration as compared with polyglutamic acid and other polyglutamic acid salts. Therefore, the calcium salt of polyglutamic acid can be used as a blood triglyceride concentration inhibitor, and can be used to produce the triglyceride concentration inhibitor. Until now, it has not been known that the calcium salt of polyglutamic acid has an action of suppressing an increase in triglyceride concentration in blood. In addition, it is not known that calcium salt of polyglutamic acid has an effect of preventing or improving hyperlipidemia or arteriosclerosis. As shown in the examples below, polyglutamic acid calcium salt increases blood triglyceride concentration, especially blood triglycerides after eating (after ingesting foods and beverages containing normal carbohydrates, lipids, proteins, etc.). An increase in concentration can be efficiently suppressed.

The calcium salt of polyglutamic acid can generally have an inhibitory effect on the increase in blood triglyceride 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)]. In addition, as an example of production of polyglutamic acid using gene recombination technology, about 9 g / L / 5 days [Ashiuchi, M., et al.] In a recombinant Bacillus subtilis ( Bacillus subtilis ISW1214 strain) introduced with a plasmid. .: 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 an increase in blood triglyceride concentration after intake of carbohydrates, lipids and / or proteins. And the inhibitory effect is remarkably excellent compared with polyglutamic acid and other polyglutamate.

  The blood triglyceride level increase inhibitor of the present invention may be the calcium salt of polyglutamic acid itself. In addition, the blood triglyceride 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. In this case, the blending amount of the polyglutamic acid calcium salt is not particularly limited, but is preferably 0.01 to 100% by mass, more preferably 0.1 to 95% by mass in the blood triglyceride concentration increase inhibitor. Preferably, 1 to 90% by mass is included, and 10 to 85% by mass is even more preferable.

  When the blood triglyceride level increase inhibitor of the present invention is used for foods, pharmaceuticals and the like, polyglutamic acid alone is administered to humans and animals in the digestive tract, intraperitoneal administration, intravascular administration, intradermal administration, subcutaneous administration, etc. In addition to various foods, pharmaceuticals, pet foods and the like. As food, in addition to general foods, the concept is to suppress the increase in blood triglyceride concentration and to reduce, prevent, improve, alleviate, and treat hyperlipidemia and arteriosclerosis risk. It can be applied to foods such as the displayed beauty food, food for the sick, and food 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 to 80% by weight, particularly preferably 0.3 to 70% by weight, and particularly preferably 1 to 60% by weight.
The effective administration (intake) amount in the above foods and preparations is preferably 0.01 g / kg body weight to 1.0 g / kg body weight per day as a calcium salt of polyglutamic acid, preferably 0.003 g / kg body weight to The weight is more preferably 0.5 g / kg body weight, and further preferably 0.01 g / kg body weight to 0.2 g / kg body weight. Moreover, the blood triglyceride concentration increase inhibitor of the present invention is effective when used before, during or after a meal, and is particularly preferably used before or during a meal, more preferably from 1 hour before the meal. preferable.

  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-described analysis method.

[Test Example] Polyglutamic acid calcium salt suppresses blood triglyceride concentration from increasing The polyglutamic acid calcium salt has a weight average molecular weight of 11,000 (prepared in Preparation Example 1) and a weight average molecular weight of 9,000 (manufactured by Meiji Food Materia Co., Ltd.) ) Of polyglutamic acid (manufactured by Meiji Food Materia Co., Ltd.) was prepared as described below.
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). A sample for oral administration was prepared so as to be 2 (w / w)% and albumin 1.0 (w / w)%, and a sample to which water was added instead of polyglutamate was prepared as a control sample. .

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, blood triglyceride concentration was measured using Triglyceride E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd., GPO / DAOS method).
Based on each measurement result of blood triglyceride concentration from 2 hours after oral administration of the sample, the difference between the maximum blood triglyceride concentration and the initial value (at the time of initial blood collection) (Δ value) is the increase in maximum triglyceride concentration. Table 2 shows the values when the control group is defined as 100.

  Based on the obtained increase in the maximum triglyceride concentration, statistically significant differences between the groups were 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 increase in the maximum triglyceride concentration can be effectively suppressed in any case where polyglutamate was administered.
Moreover, the increase in the maximum triglyceride concentration was significantly suppressed in the group administered with the calcium salt of polyglutamic acid as compared with the group administered with the sodium salt of polyglutamic acid.

  As described above, it is known that an increase in blood triglyceride concentration causes hyperlipidemia and thus arteriosclerosis. Therefore, the calcium salt of polyglutamic acid can be suitably used for the prevention and improvement of hyperlipidemia and arteriosclerosis by effectively suppressing the increase in blood triglyceride concentration.

Claims (1)

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