JP3108675B2 - Lipid metabolism improver - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the inhibition of dietary lipid absorption,
The present invention relates to a lipid metabolism-improving agent which has a postprandial hyperlipidemia-ameliorating effect and is expected to have a preventive / ameliorating effect on hyperlipidemia, arteriosclerosis, obesity and the like.

[0002]

2. Description of the Related Art In recent years, the intake of lipids has been increasing with the improvement of dietary habits or westernization. According to the National Nutrition Survey, the percentage of lipid energy in the energy intake exceeds the upper limit of the appropriate ratio (25%) since 1988,
It is steadily increasing. Especially, especially for the young generation,
The increase in animal fat intake ratio is remarkable. This constant overdose of fat can lead to obesity and elevated serum lipids, with various complications (eg,
Increase the risk of developing cardiovascular diseases, especially coronary and cerebrovascular diseases and adult diseases such as certain types of cancer such as breast cancer and colorectal cancer). It has become.

[0003] In particular, an increase in blood cholesterol concentration has been conventionally cited as a risk factor for the onset of circulatory diseases, and more recently, a blood triglyceride concentration has been regarded as a problem independent of cholesterol ( Austin, MA Am. J. Epidem. 129,249-259 (198
9), Carlson LA et al. Acta.Med. Scand. 206,351
-360 (1979), Carlson LA et al. Acta.Med. Scand.
218, 207-211 (1985), Castelli, WP Am. Heart.J. 1
12,432-437 (1986)).

[0004] Ingestion of fats and carbohydrates temporarily causes postprandial hyperlipidemia, but excessive intake leads to prolonged postprandial hyperlipidemia. Hyperlipidemia includes elevated serum cholesterol levels and elevated serum triglyceride levels,
It is well known that hypercholesterolemia is a risk factor for arteriosclerosis. In addition, in a situation where fat is consumed excessively constantly, the blood triglyceride concentration is continuously increased, and the increase in the blood triglyceride concentration may cause the onset of arteriosclerotic diseases such as hypertension and ischemic heart disease. It has been pointed out that there is a high risk of causing hypertension (Hario Nakamura, History of Medicine 157,771-775 (1991), Handbook of Hypertriglyceridemia, Shunji Ishikawa, Knowledge of Medicine 39,3-8 (198
8)).

[0005] Excessive energy due to excessive intake of lipids causes obesity, and obesity causes circulatory failure, arteriosclerosis,
Increase the incidence of adult diseases such as diabetes.

For this reason, approaches aimed at removing body fat or reducing dietary fat have been vigorously conducted.
The following methods and drugs have been proposed. First, there are a diet, a fat substitute, and an appetite suppressant as the suppression of fat intake. However, diets are often overly restricted diets, and in modern societies rich in delicious foods rich in lipids, are extremely difficult to implement in the long term. Also,
Various products have been developed as fat substitutes, but it is not easy to be satisfied with the taste of substitutes in the era of gourmet pursuit of natural taste and authenticity. More recently,
It has been noted that some fat substitutes have severe side effects. In addition, as an appetite suppressant, mazindol and fenfluramine are known, but can be administered only under the control of a physician, and their side effects have been reported (Hadler AJJ Clin. Pharm. 12,453 (1972),
Stunkar DA et al. Lanset 1,503 (1973)).

[0007] As an alternative to these difficult methods, there are drugs (nicomol, neomycin, etc.) that suppress the absorption of lipids in the gastrointestinal tract without limiting the intake of fats and the like, and the absorbed nutrients. Drugs (such as dehydroepiandrosterone) for preventing accumulation as fat have also been developed, but the occurrence of side effects is inevitable in these drugs.

[0008] In addition, dextran sulfate, which is a drug that enhances lipoprotein lipase activity in blood to promote the metabolism of blood lipid, and clofibrate and bravastatin, which are lipid metabolism improvers, are currently used. There are also concerns about the side effects of administering pharmaceuticals.

In the treatment of hypercholesterolemia,
Drugs that suppress cholesterol biosynthesis are known, but many of these drugs have side effects.

[0010] Therefore, development of a food material which is considered to have few side effects with respect to the above-mentioned drugs is expected. In food materials, it is known that various dietary fibers interfere with the absorption of cholesterol or bile acids in the digestive tract and lower blood cholesterol levels. Effective dietary fibers include pectin, guar gum, konjac mannan, seaweed polysaccharides, chitosan and the like.

[0011] In recent years, animal experiments have revealed that chitosan is particularly superior in inhibiting lipid digestion and absorption as compared with other dietary fibers. For example, it has been reported that the suppression of apparent fat absorption reaches as much as 50% when rats are fed with about 5% chitosan added to the diet (Kinari, Chemistry and Biology 34,553-557). (199
6)). However, it has been pointed out that when this intake is converted into humans per fat intake, the daily intake is as much as 15 g, and as a result of the large intake, side effects such as constipation due to strong cohesive strength may occur. In most cases, the maximum daily intake of chitosan is set at 2 g or less.

Furthermore, chitosan may inhibit fat absorption in the gastrointestinal tract and adversely affect fat-soluble vitamin absorption. In fact, it has been reported that ingestion of chitosan lowers the blood levels of vitamin A and E in the body (Kinari, supra). Since vitamin E has an effect of preventing arteriosclerosis, it is considered that such a vitamin absorption inhibitory action is a problem inherent to chitosan.

[0013] The cholesterol-lowering effect of chitosan has been reported in many test results not only in animals but also in humans, and is known (Sugano, M. et al. Nutr.
Rep. Int., 18, 531-537 (1978), Keisuke Tsuji, Food Industry 3
6,50-56 (1993), Shigehiro Hirano, Food & Food Ingredients
Journal of Japan 159, 65-71 (1994), Takashi Nishida et al.
Monthly Food Chemical 45-50 (1995), Osamu Kanauchi, supra).
However, it has been reported that chitosan significantly reduces blood cholesterol levels in humans, but has no significant inhibitory effect on blood triglyceride levels (Keiji Tsuji, supra, Nishida et al., Supra). ).

As described above, chitosan is an excellent food material as a substance that suppresses a rise in serum cholesterol concentration which is considered to be based on the absorption inhibitory action in the digestive tract, but has problems such as side effects due to large intake.

On the other hand, "a protein hydrolyzate produced by hydrolyzing a protein or a protein-containing substance with a protease or acid and having an average peptide chain length of 3 to 4 and having an effect of improving lipid metabolism," hereinafter referred to as BOREP, It has been shown to have the effect of significantly suppressing the increase in serum triglyceride concentration due to fat intake (Japanese Patent Publication No. 5-87
052 (WO89 / 06970, US08 / 608,045)).

The effects of BOREP include inhibition of lipid absorption in the gastrointestinal tract by suppressing the activity of pancreatic lipase, promotion of metabolism of triglyceride in kilomicrons by activation of lipoprotein lipase, and β-
Acceleration of oxidation, suppression of mutation from fibroblasts to preadipocytes, and preadipocytes to adipocytes, etc. have been confirmed, not only inhibiting absorption in the gastrointestinal tract, but also promoting metabolism of lipids once digested and absorbed. It is believed that multiple mechanisms of reducing blood triglyceride levels.

In addition, BOREP has no side effects (side effects due to excessive intake, inhibition of fat-soluble vitamin absorption) observed in chitosan. However, BOREP has a problem that it does not show a remarkable inhibitory effect on blood cholesterol rise as much as chitosan.

[0018]

DISCLOSURE OF THE INVENTION The present invention solves the above problems by inhibiting the digestion and absorption of lipids and improving the metabolism of the lipids once absorbed. An object of the present invention is to provide a lipid metabolism-improving agent having a prophylactic and ameliorating effect on obesity and lifestyle-related diseases such as hyperlipidemia and arteriosclerosis, which have a strong causal relationship with excessive intake.

[0019]

Means for Solving the Problems In view of the circumstances described above, the present inventors have produced a protein or a protein-containing substance by hydrolysis with a protease or an acid, and have an average peptide chain length of 3 to 4. By actually mixing and using a protein hydrolyzate (referred to as BOREP) having a lipid metabolism improving effect and chitosan for the first time, the combination drug has an effect of suppressing a blood triglyceride concentration increase and a blood cholesterol concentration increase. , Unexpectedly found a synergistic effect, further supplemented the problems seen in the single administration of each component in the dosage, etc., exhibited exceptional remarkable effects that were not seen in the single administration of each component The present invention was completed based on these. In particular, by using the mixture, the intake of chitosan can be significantly reduced.

That is, the lipid metabolism improving agent according to the present invention comprises B
It is characterized by containing a mixture of OREP and chitosan as an active ingredient.

The BOREP used in the present invention is disclosed in, for example, Japanese Patent Publication No. 5-87052 (International Publication No. WO89 / 06970) and International Publication No. WO97 / 00890, and the method described in these publications or a method analogous thereto. It can be manufactured according to The chitosan used in the present invention is known per se, and is described in detail, for example, in Chitin-Chitosan Research Society, "Last Biomass Chitin, Chitosan, 1988". Also in the present invention, an appropriate one can be selected from commercially available ones.

The blending weight ratio of BOREP and chitosan can be freely set, but is preferably from 4: 1 to 2: 3, more preferably about 3: 2, as shown in the examples described later. A high synergistic effect can be obtained at this compounding weight ratio.

The agent for improving lipid metabolism according to the present invention has an effect of inhibiting dietary lipid absorption in animals (eg, humans, rabbits, mice, etc.) and an effect of improving lipid metabolism which is considered to be based on an effect of improving postprandial hyperlipidemia. Indicates hyperlipidemia (hypertriglyceridemia, hypercholesterolemia), arteriosclerosis (atherosclerosis, cerebral atherosclerosis, coronary atherosclerosis,
It is useful as a prophylactic / ameliorating agent for hypertension and obesity. The ameliorating agent according to the present invention has a remarkable effect in preventing or ameliorating diseases associated with hypertension, such as obesity, hyperlipidemia, and the like. It is preferably used for prevention and improvement.

The lipid metabolism-improving agent according to the present invention can be used as a preparation itself (mixture of a BOREP substance and a chitosan substance) or mixed with an appropriate carrier, excipient, diluent, etc. to obtain capsules, tablets, powders and the like. And can be safely administered orally as a solid composition such as granules. These compositions can be formulated according to known methods generally used in the formulation process.

The daily dosage of the individual components may be, for example, about 45
BOREP in the range of 0-900 mg, for example, about 300
Chitosan in the range of ６００600 mg can be effectively combined. Of course, these dose ranges can be adjusted on a unit basis necessary to divide up the daily dose. Preferably, tablets as described in the Examples below are administered 6 times a day.
~ 12 tablets, most preferably 8 tablets daily (BOREP 600
mg, chitosan 400 mg).

The lipid metabolism-improving agent according to the present invention is provided not only as a preparation as described above, but also as a form containing it in foods and drinks and animal feed. This
These foods and drinks and animal feeds are used to improve lipid metabolism.
Used for purposes. Examples of the food include health foods, foods for specified health use, and general foods (chocolate, cake, ham, meat sauce, stew, milk, pudding, curry, coconut, etc.). It is preferable that the lipid metabolism improving agent according to the present invention is mixed with these foods at a ratio of 0.1 to 50%, and the improving agent is administered in a range of 750 mg to 1200 mg per day. In animal feeds, it is preferable that the improving agent is contained in a range of 0.05% to 5%. The animal feed containing the agent for improving lipid metabolism according to the present invention is useful for improving animal meat quality in the fields of livestock and fisheries by preventing the animal from becoming fat.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION BOREP which is a component of the present invention
The production method is disclosed in Japanese Patent Publication No. 5-87052, an example of which is as follows. Any protein or protein-containing substance such as animal, vegetable, or microbial sources is dispersed as a solid in water at 5 to 30% by weight, and the protease is adjusted to an optimal pH with an acid or an alkali. Add all at once or sequentially to 20-7
The protease is reacted and hydrolyzed at a temperature of 0 ° C. for 3-48 hours.

Next, the obtained protein hydrolyzate is spray-dried, or an appropriate amount of a bulking agent such as carboxymethylcellulose or dextrin is added to the protein hydrolyzate and dried and solidified to obtain a protein having an effect of improving lipid metabolism. BOREP solids, which are hydrolysates, can be obtained.

As a raw material of BOREP, any protein can be used as described above. However, as described in International Publication No. WO97 / 00890, globin proteins such as hemoglobin and myoglobin suppress increase in blood triglyceride concentration. This is particularly preferable in that the effect of performing the above can be strongly exerted.

The kind of animal that is the source of such a globin protein is not particularly limited, and it may be cow, pig,
Blood from sheep, humans, horses, etc. can be used widely.

As the hydrolase to be used, for example, one or more of acidic protease, neutral protease and alkaline protease can be used.

The BO obtained by the hydrolysis as described above
As an active ingredient of REP, several oligopeptides have been confirmed. For example, in International Application No. WO97 / 00890,
Among the components of BOREP, in particular, Leu-Val-Val-Ty
Three peptides of r-Pro-Trp-Thr, Val-Thr-Leu and Val-Tyr-Pro, and in WO97 / 35875, Val-Val-Tyr-Pro
All have been confirmed to be excellent in the action of suppressing the increase in blood triglyceride concentration.

Chitosan, another component of the present invention, is a known substance as described above, and is described in detail, for example, in "The Last Biomass Chitin, Chitosan, 1988" edited by Chitin and Chitosan Research Society. . Also in the present invention, an appropriate one can be selected from commercially available ones.

[0034]

EXAMPLES Example 1: Inhibitory effect on increase in serum lipid concentration (experiment using mice)

Adapted ICR male mice (6-7) preliminarily reared on solid feed (CE-2, CLEA Japan)
6 weeks (about 33 g) were randomly selected into 6 groups (BOREP administration group, chitosan administration group, and hereafter referred to as the product of the present invention B)
A mixture of OREP and chitosan (BOREP and chitosan combination ratio 4: 1, 3: 1, 3: 2, 2: 3) administration group)
Used separately. After an overnight fast, 250 mg of olive oil supplemented with 5% cholesterol and the above-mentioned administration were orally administered to each group, and two hours after administration, serum triglyceride concentration and serum cholesterol concentration were measured by an enzyme method. The measurement was performed using "Triglyceride E-test" and "Cholesterol E-test" manufactured by Wako Pure Chemical Industries, Ltd., according to the protocol specified in the kit.

From the measured concentration values, a 50% inhibition rate (ID50 mg / mouse) of the inhibitory effect on the increase in serum lipid concentration in each administered substance.
(Measured values in the table) are shown in Tables 1 and 2 below. The ratio (B: C) in the table is the BORE in the product of the present invention.
P: Indicates the compounding ratio of chitosan. ID50 of the product of the present invention (in the table
(b)) is a theoretical value assuming an additive effect obtained by adding the values obtained by multiplying the measured values of ID50 of BOREP and chitosan alone by their respective mixing ratios. The synergistic effect is expressed as a percentage of the measured value with respect to the calculated value of the product of the present invention. The smaller this value, the greater the synergistic effect of the combination of the two components.

[0037]

[Table 1]

[0038]

[Table 2]

In order to visually grasp and examine the data in Tables 1 and 2, the measured ID50 values were plotted in FIGS. 1 and 2, respectively. The straight line in the figure connecting the ID50 of BOREP and chitosan alone is a continuous plot of the calculated value (theoretical value) of the additive effect. The measured values of ID50 of the product of the present invention (plots indicated by open squares in the figure) were plotted on the origin side with respect to this straight line, and it was confirmed that there was a synergistic effect at any mixing ratio.

According to this experiment, the combination of BOREP and chitosan has an unexpectedly strong synergistic effect on the inhibitory effects on the increase in triglyceride concentration and cholesterol concentration as compared to the effect expected when each component is simply mixed. It became clear that it exhibited.

Example 2 Inhibitory Effect on Serum Lipid Concentration Increase (Experiment Using Rabbit)

Sixteen randomized NZW male rabbits (7 weeks old, about 3 to 4 kg) which had been preliminarily reared on a solid feed (CR-1, Nippon Clea Co., Ltd.) (control group,
They were divided into a BOREP administration group, a chitosan administration group, and a product of the present invention (a BOREP / chitosan combination ratio of 3: 2) administration group.

The control group was bred for 2 weeks on a diet supplemented with 1% cholesterol. The other three groups are the cholesterol 1
The feed was bred for 2 weeks on a feed mixture containing 5% of each of the administered substances. At the beginning of this two week breeding period,
Blood is collected from the auricular vein after 1 week and 2 weeks (0.
5 to 1 ml), and the serum triglyceride concentration and serum cholesterol concentration were measured by an enzyme method. The measurement was performed using "Triglyceride E-test" and "Cholesterol E-test" manufactured by Wako Pure Chemical. The results are shown in Tables 3 and 4. The effect of each administration group on the control group (a in the table) and the effect on the present invention group (b in the table) were examined by a significant difference test method (Fisher's PLSD method).
The difference in the case of <0.05 was determined to be significant.

[0044]

[Table 3]

[0045]

[Table 4]

As compared with the control group, the serum triglyceride concentration and the serum cholesterol concentration of the group to which BOREP, chitosan and the product of the present invention were administered were both reduced. BO
REP was known to significantly suppress the increase in serum triglyceride concentration due to fat ingestion, but it was revealed that repeated ingestion showed an inhibitory effect not only on serum triglyceride concentration but also on cholesterol concentration.

Further, as is clear from the results after 2 weeks of rearing, the product of the present invention has a stronger inhibitory effect on both serum triglyceride and serum cholesterol concentration than the single administration of the combined components BOREP and chitosan. Indicated. From these results, the product of the present invention was found to be BOREP,
It was shown to be useful as an inhibitor of postprandial hyperlipidemia (hypertriglyceridemia, hypercholesterolemia) as compared to chitosan alone.

Example 3: Inhibitory effect on increase in serum lipid concentration (human test)

[0049] Adult volunteers (12 males, ages 27-50 years) with serum triglyceride or cholesterol levels at levels from the normal to the border region were used. The test was conducted in accordance with the Helsinki Declaration, and the subjects signed the consent form at their own volition after receiving sufficient explanation about the test. The test was performed by the crossover method, and the rest period between tests was one week.

The outline of the test method is as follows. The subjects fasted for 12 hours or more from the night before, and ate omelets cooked with 5 chicken eggs and 50 g of butter at 9 am the following morning. After eating,
BOREP administration group, chitosan administration group, the product of the present invention (BOR
The administration group ingested a tablet containing 1 g of each component (in the administration group of the present invention, 600 mg of BOREP and 400 m of chitosan).
g). For the control group, tablets not containing the above components were ingested. Table 5 shows the nutritional components of the ingested omelet.

[0051]

[Table 5]

Before meal (0 hour), after meal 1,2,3,4
Blood was collected from the brachial vein at 5 or 6 hours, the serum was separated, and the concentrations of triglyceride and total cholesterol were measured by the enzyme method. The measurement was performed using "Triglyceride E-test" and "Cholesterol E-test" manufactured by Wako Pure Chemical. FIG. 3 shows the change over time in the serum triglyceride concentration, and FIG. 5 shows the change over time in the serum cholesterol concentration.

As shown in FIG. 3 and FIG. 5, the serum triglyceride concentration and the serum cholesterol concentration were increased by ingesting the fat diet, and the increase was suppressed by administering BOREP, chitosan and the product of the present invention. In the figure, * represents the risk factor P <0.05, and ** represents the risk factor P.
<0.01, the group treated with the product of the present invention was a control group, BOREP
It indicates that there is a significant difference between the administration group and the chitosan administration group. As can be understood from FIGS. 3 and 5, administration of the product of the present invention significantly suppressed increase in serum triglyceride concentration and increase in serum cholesterol concentration after fat load, as compared to administration of BOREP and chitosan alone.

FIGS. 4 and 6 show the serum triglyceride concentration and serum cholesterol concentration, respectively, of 0-6.
AUC (area under serum compound concentration time curve) of time is shown. As is clear from these graphs, the inhibitory effect of the administration of the product of the present invention was stronger than that of the administration of BOREP or chitosan alone, for both the increase in serum triglyceride and the increase in serum cholesterol.

Example 4: Composition of tablet

One example of the tablet composition of the lipid metabolism improving agent according to the present invention is shown below.

BOREP 75mg, Chitosan 50m
g, corn starch 10 mg, crystalline cellulose 20
mg, lactose appropriate amount, silicon dioxide 2 mg, sucrose fatty acid ester 5 mg, and more as tablets (tablet weight 2
15 mg).

As can be understood from the above examples, BOREP, which is particularly effective in suppressing an increase in serum triglyceride concentration,
The lipid metabolism improving agent of the present invention, which is combined with chitosan, which is particularly effective for suppressing a rise in serum cholesterol concentration,
An unexpectedly strong effect (synergistic effect) was obtained for both the increase in serum triglyceride concentration and the increase in serum cholesterol concentration, as compared with the effect of using the above two components alone.
The lipid metabolism-improving agent of the present invention can significantly reduce the dose when the above components are used alone, and as a result, exerts an effect on humans with reasonable intake, especially It is useful as a prophylactic / ameliorating agent for hyperlipidemia, arteriosclerosis and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the synergistic effect of the combination of BOREP and chitosan on the inhibitory effect on an increase in serum triglyceride concentration after fat loading.

FIG. 2 is a graph showing the synergistic effect of the combination of BOREP and chitosan on the inhibitory effect on the increase in serum cholesterol concentration after fat loading.

FIG. 3 is a graph of a time-course study of the effect of the invention on serum triglyceride concentration after fat loading.

FIG. 4. Serum triglyceride increase after fat loading (A
7 is a graph of the effect of the invention on UC0-6h).

FIG. 5 is a graph of a time-course study of the effect of the invention product on serum cholesterol concentration after fat loading.

FIG. 6: Serum cholesterol increase after fat loading (A
7 is a graph of the effect of the invention on UC0-6h).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61K 31/722 A61K 31/722 A61P 3/06 A61P 3/06 (72) Inventor Chisuko Fukuhama 180 Koecho, Ikeda-shi, Osaka Hankyu Kyoei Bussan Co., Ltd. Pharmaceutical Research Laboratories (72) Inventor Hiroaki Fujino 180 Kouecho, Ikeda-shi, Osaka Hankyu Kyoei Bussan Co., Ltd. Pharmacological Research Laboratories (56) References JP-A 7-194316 (JP, A) International Publication 89 / 6970 (WO, A1) WO 97/890 (WO, A1) NUTRITION REPORTS INTERNALATION, 1978, Vol. 18, No. 9, pp. 531- 537 Monthly Food Chemical, 1995-2, pp. 45-50 Food Industry, 1993, Vol. 36, pp. 50-56 FOODS AND FOOD IN GREDIENTS JOURNAL OF JAPAN, 1994, No. 159, pp. 65-71 (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 38/01 A23K 1/16 A23L 1/30 A23L 1/305 A61K 31/722 A61P 3/06 CA (STN)

Claims (8)

(57) [Claims] 1. An active ingredient comprising a protein hydrolyzate produced by hydrolyzing a protein or a protein-containing substance with a protease or an acid and having an effect of improving lipid metabolism having an average peptide chain length of 3 to 4, and chitosan. An agent for improving lipid metabolism, characterized in that: 2. The lipid metabolism improving agent according to claim 1, wherein the weight ratio of the protein hydrolyzate to chitosan is from 4: 1 to 2: 3. 3. The lipid metabolism improving agent according to claim 1, wherein the weight ratio of the protein hydrolyzate to chitosan is 3: 2. 4. A dietary lipid absorption inhibitor.
The lipid metabolism improving agent according to any one of the above. 5. A method for improving postprandial hyperlipidemia.
The lipid metabolism improving agent according to any one of the above. 6. The lipid metabolism improving agent according to any one of claims 1 to 3, which is a preventive / ameliorating agent for hyperlipidemia, arteriosclerosis, hypertension, and obesity. 7. A food or drink for improving lipid metabolism, comprising the lipid metabolism improving agent according to any one of claims 1 to 3. 8. An animal feed for improving lipid metabolism, comprising the lipid metabolism improving agent according to any one of claims 1 to 3.