JP2012144484A - 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 potassium 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 hypertriglyceridemia and hyperlipidemia are 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 reduces, prevents, improves, alleviates or treats the risk of developing hypertriglyceridemia, hyperlipidemia and arteriosclerosis. It is an object of the present invention to provide a blood triglyceride level increase inhibitor useful for food use that is a pharmaceutical use or non-pharmaceutical use.

  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 salts of polyglutamic acid, potassium 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 potassium 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, ameliorates, treats, reduces the risk of developing hypertriglyceridemia and hyperlipidemia, Is useful for reducing, preventing, improving, mitigating and treating the risk of developing arteriosclerosis.

Hereinafter, the present invention will be described in detail.
The blood triglyceride concentration inhibitor of the present invention contains a potassium 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 potassium 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 above structural formula are substituted with potassium. The potassium 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 potassium, 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 with potassium, but substantially all of the carboxyl groups are substituted with potassium. Particularly preferred. Moreover, it is preferable that the carboxyl group which will be located in the terminal in the said structural formula is also substituted with potassium.
The potassium 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 potassium 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 potassium salt of polyglutamic acid has the effect of suppressing the increase in triglyceride concentration in the blood. In addition, it is not known that potassium salt of polyglutamic acid has an effect of preventing or improving hyperlipidemia or arteriosclerosis. As shown in the examples below, polyglutamic acid potassium salt increases blood triglyceride concentration, especially blood triglyceride after meals (after ingesting foods and beverages containing normal carbohydrates, lipids, proteins, etc.). An increase in concentration can be efficiently suppressed.

The potassium salt of polyglutamic acid generally has an inhibitory effect on the increase in blood triglyceride concentration regardless of its molecular weight, but tends to be more effective on the low molecular weight side.
The potassium salt of polyglutamic acid used in the present invention preferably has a weight average molecular weight of 500 or more, more preferably 1000 or more, further preferably 2,000 or more, and 3,000 or more. More preferably, it is particularly preferably 5,000 or more.
The upper limit of the weight average molecular weight of the potassium salt of polyglutamic acid to be used is preferably about 5,000,000, but when the preparation is used and the blood triglyceride concentration inhibitor of the present invention is used as an oral liquid preparation From the standpoints of throat sensation, sliminess, ease of swallowing, etc., its viscosity is preferably relatively low, and the weight average molecular weight of the potassium salt of polyglutamic acid used is 1,000,000 or less Is more preferably 500,000 or less.
More specifically, the weight average molecular weight of the potassium salt of polyglutamic acid used in the present invention is preferably 500 to 5,000,000, more preferably 1,000 to 1,000,000. More preferably from 3,000 to 500,000, further preferably from 3,000 to 500,000, particularly preferably from 4,000 to 400,000, and from 5,000 to 300,000. Particularly preferred.
The weight average molecular weight can be measured, for example, by high performance liquid chromatography using a gel filtration column.

The potassium 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 Examples below. It can be obtained by neutralizing with an aqueous potassium oxide solution. It can also be produced by microorganisms cultured in a mixed medium containing potassium. The optical activity of glutamic acid constituting the potassium 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 potassium 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 potassium salt of polyglutamic acid itself. In addition, the blood triglyceride level 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 potassium salt of polyglutamic acid. May be included. In this case, the blending amount of the potassium salt of polyglutamic acid is not particularly limited, but it 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 5 to 85% by mass is particularly 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 foods, in addition to general foods, the concept is to suppress the rise in blood triglyceride concentration, reduce, prevent, improve, alleviate, and treat hypertriglyceridemia, hyperlipidemia and arteriosclerosis risk. Accordingly, the present invention 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, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, a corrigent and the like were added to the potassium salt of polyglutamic acid as necessary. 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 potassium 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 each of the above foods and preparations is preferably 0.01 g / kg body weight to 1.0 g / kg body weight per day as a potassium salt of polyglutamic acid, preferably 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 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.
The subject of administration or ingestion is not particularly limited as long as it is a person who needs it, but is preferably a person having a fasting blood triglyceride level of 100 mg / dL or more.

  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 method and weight average molecular weight determination method-1 of polyglutamic acid potassium salt:
The quantitative determination of the potassium salt of polyglutamic acid and the measurement of the weight average molecular weight were carried out by gel filtration using a D-6000 (manufactured by Hitachi High-Technologies Corporation) HPLC system. The analysis conditions were TSKGel G4000PWXL and TSKGelG6000PWXL gel filtration columns (trade name, manufactured by Tosoh Corporation) as the analysis column, 0.1M sodium sulfate as the eluent, a flow rate of 1.0 mL / min, and a column temperature of 50 ° C. The UV detection wavelength was 210 nm. The concentration was calculated by preparing a calibration curve using polyglutamic acid having a molecular weight of 880 k (manufactured by Meiji Food Materia Co., Ltd.) as a standard product. In addition, the weight average molecular weight was determined by using pullulan (trade name: Shodex STANDARD P-82, manufactured by Showa Denko KK) in advance, and a plurality of polyglutamic acids having different molecular weights (trade name, manufactured by Wako Pure Chemical Industries, Ltd. (trade name). : 1622-1411, 162-21401), manufactured by SIGMA-ALDRICH (trade names: P-4886, P-4761), manufactured by Meiji Food Materia Co., Ltd. (molecular weight: 880 k)).

Method of measuring weight average molecular weight of potassium salt of polyglutamic acid-2:
The weight average molecular weight measurement of the potassium salt of low molecular weight polyglutamic acid having a molecular weight of about 10 k was performed 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.

Metal analysis of polyglutamate:
Metal analysis of polyglutamate was performed by ion chromatography using a D-7000 (manufactured by Hitachi High-Technologies Corporation) HPLC system. Analytical conditions used were Shodex IC YK-G for the guard column, Shodex IC YK-421 (both trade names, manufactured by Showa Denko KK) for the analytical column, an eluent of 1.5 mM citric acid aqueous solution, and a flow rate of 1.0 mL. Detection was performed using an electric conductivity detector at a column temperature of 40 ° C./min. Using a sodium standard solution (1000 ppm) and a potassium standard solution (1000 ppm) manufactured by Kanto Chemical Co., Ltd. as standard samples, a calibration curve is prepared in the range of 10 to 100 mg / L for these standard products, and a polybasic curve is prepared based on these calibration curves. Metal analysis of glutamate was performed.

[Preparation Example 1] Preparation of potassium salt of polyglutamic acid having a weight average molecular weight of 12,000 10% (w / w) of sodium salt of polyglutamic acid having a weight average molecular weight of 9,000 (manufactured by Meiji Food Materia Co., Ltd.) 500 mL of an aqueous solution was prepared and adjusted to pH 2 or lower using hydrochloric acid under ice cooling. 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 an aqueous potassium hydroxide solution so that the pH became 7 or more. This acid treatment and neutralization treatment with potassium 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 14,000 rpm for 5 minutes (same as above), and the collected sample was dried under reduced pressure to obtain 27.4 g of a solid. The weight average molecular weight of this sample was calculated to be 12,000 by the aforementioned analysis method. In addition, in this sample, an amount of potassium corresponding to the amount of carboxyl groups of polyglutamic acid was detected, and sodium was below the detection limit. Therefore, substantially all the hydrogen atoms of the carboxyl groups of polyglutamic acid before neutralization were converted to potassium. It was confirmed that it was replaced.

[Preparation Example 2] Preparation of potassium salt of polyglutamic acid having a weight average molecular weight of 240,000 5% (w / w) of sodium salt of polyglutamic acid having a weight average molecular weight of 350,000 (manufactured by Meiji Food Materia Co., Ltd.) 1 L of an aqueous solution was prepared and adjusted to pH 1 or lower using hydrochloric acid under ice cooling. 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 this washing operation was repeated twice, the resulting precipitate was suspended in 800 mL of distilled water and neutralized with an aqueous potassium hydroxide solution so that the pH was 7 or more. This acid treatment and neutralization treatment with potassium hydroxide are performed again, and the precipitate formed by the addition of ethanol is collected by centrifugation at 14,000 rpm for 5 minutes (same as above), and the collected sample is subjected to vacuum drying. As a result, 36.2 g of a solid substance was obtained. The weight average molecular weight of this sample was calculated to be 240,000 by the above analysis method. In addition, in this sample, an amount of potassium corresponding to the amount of carboxyl groups of polyglutamic acid was detected, and sodium was below the detection limit. Therefore, substantially all the hydrogen atoms of the carboxyl groups of polyglutamic acid before neutralization were converted to potassium. It was confirmed that it was replaced.

[Preparation Example 3] Preparation of potassium salt of polyglutamic acid having a weight average molecular weight of 6,000 20% (w / w) of sodium salt of polyglutamic acid having a weight average molecular weight of 9,000 (manufactured by Meiji Food Materia Co., Ltd.) 125 mL of an aqueous solution was prepared and adjusted to pH 1 or lower using hydrochloric acid. Subsequently, the obtained PGA solution was incubated at 95 ° C. for 12 hours, and the resulting acid precipitate was centrifuged at 8,000 rpm for 5 minutes (trade name: himacCR21GIII, manufactured by Hitachi Koki Co., Ltd.). The collected precipitate was washed with the same amount of distilled water and again subjected to centrifugation. A precipitate obtained by repeating this washing operation twice was suspended in 300 mL of distilled water, and neutralized with an aqueous potassium hydroxide solution so that the pH became 7 or more. This acid treatment and neutralization treatment with potassium 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 14,000 rpm for 5 minutes (same as above), and the collected sample was dried under reduced pressure to obtain 16.5 g of a solid. The weight average molecular weight of this sample was calculated to be 6,000 by the above analysis method.

[Test Example 1] Inhibition of blood triglyceride concentration increase by potassium salt of polyglutamic acid Polyglutamic acid potassium salt having a weight average molecular weight of 12,000 and 240,000 (prepared in Preparation Examples 1 and 2) and a weight average molecular weight of 9, Samples for oral administration were prepared using polyglutamic acid sodium salt (Meiji Food Materia Co., Ltd.) of 000 and 350,000 (Meiji Food Materia Co., Ltd.) as follows.
In addition, an oral administration test was performed as follows using 10-week-old male mice (C57BL / 6J Jcl: manufactured by CLEA Japan, Inc.) of 10 mice per group.

[Test Example 2] Inhibition of blood triglyceride concentration increase by low molecular weight potassium polyglutamic acid potassium salt Polyglutamic acid potassium salt having a weight average molecular weight of 6,000 (prepared in Preparation Example 3) and poly (1) having a weight average molecular weight of 12,000. A sample for oral administration was prepared as follows using a potassium salt of glutamic acid (prepared in Preparation Example 1) and a sodium salt of polyglutamic acid having a weight average molecular weight of 9,000 (manufactured by Meiji Food Materia Co., Ltd.).
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. The values when the control group is defined as 100 are shown in the following Table 2 (result of Test Example 1) and Table 3 (Result of Test Example 2).

  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 a sodium salt administration group of similar molecular weight. And the potassium salt-administered group were tested, 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. This maximum triglyceride concentration inhibitory effect was particularly excellent when a relatively low molecular weight compound having a weight average molecular weight lower than 15,000 was used.
In addition, administration of polyglutamic acid potassium salt showed a markedly superior blood triglyceride concentration-inhibiting effect compared to administration of polyglutamic acid sodium salt. This is because the inhibitory effect over the maximum triglyceride concentration increase inhibitory effect obtained by using a sodium salt of polyglutamic acid having a weight average molecular weight of 9,000 is 2.5 times that of the weight average molecular weight of 240,000. It can also be understood from the fact that it is achieved by the potassium salt of polyglutamic acid.

From the results in Table 3, it can be reconfirmed that administration of polyglutamic acid potassium salt has a markedly superior blood triglyceride concentration-inhibiting effect as compared to administration of polyglutamic acid sodium salt.
It was also revealed that the effect of suppressing the increase in blood triglyceride concentration of polyglutamic acid potassium salt having an average weight molecular weight of 6,000 is equivalent to that of polyglutamic acid potassium salt having an average weight molecular weight of 12,000.

  As described above, it is known that an increase in blood triglyceride concentration causes hyperlipidemia and thus arteriosclerosis. Therefore, the potassium 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 potassium salt of polyglutamic acid as an active ingredient.