JP2003137805A - Serum lipid-reducing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a natural material-derived serum lipid-reducing agent having an excellent serum lipid-improving activity and less adverse effect, and a health food. SOLUTION: This serum lipid-reducing agent is obtained by incorporating a phycobili protein extracted from algae as an active ingredient, the serum lipid-reducing agent containing phycocyanin as an active ingredient and the health food containing phycocyanin as the active ingredient are also provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a phycocyanin which is a phycobiliprotein extracted from algae, a phycobiliprotein extracted from algae of the genus Spirulina, or a natural lipid-derived serum lipid-lowering agent containing phycocyanin as an active ingredient and having few side effects. Regarding health food as an ingredient.

[0002]

2. Description of the Related Art In recent years, westernization of eating habits has improved the physical condition and nutrition of Japanese people, but on the other hand, it has also increased the number of lifestyle-related diseases. Obesity, hyperlipidemia, and hypercholesterolemia cause diseases such as arteriosclerosis, cerebral thrombosis, and ischemic heart disease. Mortality rates due to these lifestyle-related diseases are always high, and drug therapy is used for the treatment. However, although drug treatment is highly effective,
There is a risk of causing disorders due to side effects, and attempts have been made to improve these lifestyle-related diseases by using food components that are commonly taken in daily life.

For example, soybean protein (SBP) has a low lysine (Lys) / arginine (Arg) ratio (1.0 or less) among the amino acids constituting soybean protein, and a sulfur-containing amino acid (M).
et, Cys) content is high, it has been reported that it has the effect of promoting the extracorporeal excretion of neutral steroids contained in the diet and bile acids secreted into the intestinal tract to lower serum cholesterol levels. It is recognized by the Ministry of Health, Labor and Welfare as a food material for specified health use (supervised "foods with high cholesterol" by the Ministry of Health, Labor and Welfare Bureau of Health and Sanitation, Food and Health Division, Newly Developed Food and Health Measures Office)

Health foods have been further advanced in attempts to prevent or treat lifestyle-related diseases by using the food components that are normally taken in daily life. Although health foods are not as prominent as drug therapy, they have the characteristics of further improving the preventive or curative effects of lifestyle-related diseases by food ingredients, and are less sensitive to side effects than drug therapy, and health foods have already formed a large market in modern society. is doing.

Under these circumstances, a serum lipid-lowering agent and a health food derived from natural products having excellent preventive or therapeutic effects on hyperlipidemia and hypercholesterolemia and having few side effects are desired.

[0006]

The problem to be solved by the present invention is to provide a serum lipid-lowering agent derived from a natural product and a health food which have an excellent serum lipid-improving effect and have few side effects.

[0007]

[Means for Solving the Problems] The present inventors have investigated components derived from various natural products and found that edible algae have an excellent serum lipid-improving action without side effects. It was found that red algae have a high serum lipid-improving effect, and that phycobiliprotein among various components contained in these algae, especially phycocyanin extracted from the cyanobacterium Spirulina, has a higher serum lipid-improving effect than soybean protein. As a result, the present invention has been completed. That is, the present invention provides a serum lipid lowering agent containing phycobiliprotein extracted from algae as an active ingredient, and a serum lipid lowering agent containing phycocyanin as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The phycobiliprotein extracted from algae used in the present invention is contained in algae such as cyanobacteria, red algae, and cryptoalgae. Phycocyanin, which is one of the phycobiliproteins, is particularly abundant in cyanobacteria. As cyanobacteria, Spirulina genus, Fischerella genus, Nostoc genus, Cynechocystis genus, Cynechococ
The genus cus), the genus Tolypothrix, etc. are mentioned, and the genus Spirulina, which has been used for food and whose safety has been confirmed, is particularly preferable. As the health food, algal cells themselves may be used, but it is more preferable to extract phycocyanin in the algal cells and to purify the phycocyanin to be used in a state where there are few impurities from the viewpoint of the effect.

The phycocyanin used in the present invention is extracted from the above-mentioned algae with water or a buffer solution, centrifuged to remove insoluble matter, and ammonium sulfate is added to the aqueous solution and centrifuged.
After dialysis, it can be obtained by purification with DEAE cellulose. Phycoerythrin or allophycocyanin can also be obtained using the same purification procedure using red algae or cyanobacteria.

Among these, phycocyanin, which is obtained from spirulina of cyanobacteria, which is an alga which is produced on an industrial scale and whose safety has been confirmed and is used for food, and which is used as an edible pigment, is most preferable.

There are already many reports on the safety of phycocyanin. For example, the acute toxicity test
Oral, LD ₅₀ is 30 g / kg or more (corporate body)
Japan Science Feed Association, Chronic toxicity test (SD rat, feed containing 1% phycocyanin composition, oral administration for 12 months, determination result of no chronic toxicity factor (Saitama Medical University, Professor Takemoto).

Mutagenicity test (for histidine requirement
Salmonella typhimurium TA97, TA98, TA10
0, TA102 strain, S9 at a concentration of 0.1-10 mg / plate (regardless of the presence or absence of a metabolic activation promoter, no increase in the number of revertant colonies was observed in 4 strains, and mutagenicity was negative ( Tokyo Metropolitan Institute of Health Annual Report 47, Supplement, 1996
Year). Goto et al. Have also reported in human studies that spirulina has no side effects (NUT. REP. INTERNAT
IONAL, Volume 37, 6, 1988, 1329-1337).

The purity of the phycocyanin used in these safety tests is the absorbance at 620 nm and 280 nm described later.
The analytical method calculated from the ratio of the absorbances is 60%. Other mixed components are considered to be phycobiliproteins and proteins other than phycocyanin, but even this level of purity has sufficient safety.

The phycobiliprotein of the present invention, particularly the serum lipid lowering agent containing phycocyanin as an active ingredient, is preferably orally administered. Such preparations include tablets, capsules, granules, fine granules, powders, troches, injections, and
It can be prepared in the form of preparations such as emulsions, suspensions and syrups. Phycobiliprotein, for example, phycocyanin is a water-soluble protein that is hygroscopic, easily dissolves in water and exhibits a bright blue color, and has low stability to light. And sugar-coated tablets are particularly preferable. The shape and administration method of the health food may be any shape and administration method according to the serum lipid lowering agent.

In the preparation of these, for example, non-toxic excipients, binders, disintegrating agents, lubricants, antioxidants, isotonic agents, buffers, coating agents which are usually used for this kind of drug. , A flavoring agent, a solubilizing agent, a base material, a dispersant, a stabilizer, a colorant and the like can be used for formulation by a known method. The content of the phycobiliprotein, which is the active ingredient of the present invention, such as phycocyanin, in such a formulation varies depending on the dosage form, but is generally 1
It is desirable to contain at a concentration of ~ 100% by mass,
In addition, the purity of phycocyanin was determined by measuring the absorbance at 620 nm and 2
It is preferably 80% or more by the analytical method calculated from the ratio of the absorbance at 80 nm. In the case of health food, the purity of phycocyanin may be 60% or more.

The dose of the serum lipid lowering agent or health food of the present invention can be varied over a wide range depending on the severity of symptoms, the judgment of a doctor, etc. Generally, it is orally administered as an active ingredient per day per kg of body weight. For example, phycocyanin 0.
It is preferable to administer 5 to 50 mg, preferably 0.5 to 20 mg. In addition, the above dose can be administered once or several times a day. These can be appropriately changed according to the severity of the patient's symptoms and the diagnosis of the doctor.

The serum lipid lowering agent or health food containing the phycobiliprotein of the present invention, in particular, phycocyanin as an active ingredient suppresses absorption of cholesterol from the intestinal tract, promotes excretion, and improves serum lipids.
It is effective in the prevention and treatment of hyperlipidemia, arteriosclerosis, ischemic heart disease and stroke.

[0018]

EXAMPLES Examples of the present invention will be specifically described below, but the present invention is not limited to the following examples.

(Production Example of Phycocyanin) 500 g of Spirulina powder was added to 7500 ml of a phosphate buffer solution (pH 6.
It was suspended in 0), stirred for 1 hour, and then extracted in an incubator at 30 ° C. for 16 hours. The extract was centrifuged twice and the supernatant was collected. Further, the supernatant was salted out with a 20% ammonium sulfate solution, centrifuged, and then the supernatant was further salted out with a 50% ammonium sulfate solution overnight. This solution was centrifuged to obtain a blue precipitate. The precipitate was dialyzed against ion-exchanged water, dialyzed against distilled water again, and then freeze-dried to obtain 80 g of crude phycocyanin.

Next, 20 g per 16 g of crude phycocyanin
Crude phycocyanin was purified using DEAE anion exchange cellulose pre-equilibrated with 0 g of 500 mM potassium phosphate buffer to give 3.82 g of phycocyanin. Phycocyanin (100 mg) was dissolved in phosphate buffer (pH 6.6) to make 100 ml, and the solution was diluted 10 times to obtain a solution at 620 nm (phycocyanin absorption maximum wavelength) and 280 nm (protein-derived absorption wavelength). When the ratio of absorbance was measured, it was A ₆₂₀ / A ₂₈₀
= 3.6, and the purity was 80%.

[0021] Since the _A 620 _{/ A 280} of phycocyanin is 4.5, the purity of phycocyanin is calculated by the following equation. (Kazutoshi Nishizawa et al., 497 pages, Algae Research Method, 1979, Kyoritsu Publisher) Phycocyanin Purity (%) = 3.6 / 4.5 × 100 =
80%

[0022] (Example 1) Effect on cholesterol absorption in Caco-2 cell culture ^{[4- 14} C] - cholesterol 0.1 [mu] Ci / ml,
An emulsion containing 0.1 mM cholesterol, 1 mM oleic acid, 0.5 mM monoolein, 0.6 mM L-α-phosphatidylcholine, and 6.6 mM taurocholic acid was prepared, and this was used as solution 1 to measure cholesterol absorption. . Intestinal C cultured in a 48-well plate
The medium (DMEM + 10% FCS) was removed by suction from the aco-2 cells.

0.2 ml of the above-prepared solution 1 was dispensed into each well, and a phycocyanin crude extract and phycocyanin were used as test sections, and a caseintrypsin hydrolyzate (negative control) and soybean protein were used as control sections. Add 0.2 mg each of pepsin hydrolyzate (positive control), add 5% CO ₂ , 3
Incubated at 7 ° C for 20 minutes.

After that, the solution 1 was removed by suction to obtain 0.5 ml.
It was washed twice with DMEM. 0.5m of 0.1% SDS solution
The cells were detached by adding 1 / well and transferred to a vial.
The Aquasol-2 were absorbed with 7.5ml added liquid scintillation counter here ^{[4- 14} C] - to measure cholesterol. The results are shown in Table 1.

[0025]

[Table 1] (Note) Significant difference between different signs (p <0.05)

From the results shown in Table 1, both phycocyanin crude extract and phycocyanin significantly inhibited cholesterol absorption in Caco-2 cells as compared with casein trypsin hydrolyzate, and the inhibitory effect was the soybean protein pepsin hydrolyzate. Was equal to or higher than.

The effects on cholesterol micelle solubility (Example ^{2) In vitro [4- 14 C} ] - cholesterol 0.1 [mu] Ci / ml,
An emulsion containing 0.1 mM cholesterol, 1 mM oleic acid, 0.5 mM monoolein, 0.6 mM L-α-phosphatidylcholine, and 6.6 mM taurocholic acid was prepared, and the emulsion stability was measured.

To the above solution, a phycocyanin crude extract and a phycocyanin purified product were added as test sections, and casein trypsin hydrolyzate (negative control) and soybean protein pepsin hydrolyzate (positive control) were added as control sections, and the mixture was added to a vortex mixer. And stirred, and then sonicated. The mixture was incubated at 37 ° C for 24 hours with shaking to give a micelle solution. From this solution, 50 μl was taken in a vial, 10 ml of an emulsion scintillator was added, and the radioactivity was measured to measure the micelle solubility. The results are shown in Table 2. The additive-free system was 100%.

[0029]

[Table 2] (Note) Significant difference between different signs (p <0.05)

From the results shown in Table 2, both the phycocyanin crude extract and the phycocyanin significantly suppressed the cholesterol micelle solubility in vitro as compared with the casein trypsin hydrolyzate.

(Example 3) Serum cholesterol lowering effect of phycocyanin (animal experiment) Male Wistar rats (body weight: 90 to 110 g) were bred for 5 days with a basic feed for breeding, and after fasting for 1 day, a basic diet (casein 23. 25%, lard 5%, corn oil 1%, cellulose 5%, minerals 3.5%, vitamins 1%, sugar 2
0.1%, starch 40.2%, cholesterol 0.5%,
Sodium cholate 0.25%, Choline chloride 0.2%)
Was given as a control group, 3% Spirulina was added to this basic diet (3% Sp group), 3% phycocyanin was added (3% PC group), and the phycocyanin extraction residue was 3 % Group (3% Re group) was set up and 6 animals per group were bred for 4 days (the components in the added sample were adjusted with sugar and starch).

Then, serum lipids (total cholesterol level,
HDL-cholesterol level, LDL + VLDL-cholesterol level) are analyzed and the arteriosclerosis index (Atherogenic Index) is H
It was calculated by the formula DL-c / serum TC. The results are shown in Table 3. The unit of numbers in the table is mg / dl, and there is a significant difference between different signs (* p <0.05, ** p <0.01, *** p <0.001).
Serum TC in the table is serum total cholesterol level, HDL-c is HD.
Cholesterol level in L (high density lipoprotein), LDL + VL
DL-c represents the sum of the cholesterol of LDL (low density lipoprotein) and VLDL (ultra low density lipoprotein).

Comparative Example Cholesterol-lowering Action of Soybean Protein (SBP) The same test was carried out on soybean protein whose cholesterol-lowering action was confirmed, and the results are also shown in Table 3.

[0034]

[Table 3]

From the results of Table 3, the effect of suppressing the increase in serum total cholesterol level was the most effective in the phycocyanin group (p <
0.01), LDL + is said to be bad cholesterol
The VLDL-cholesterol level was also the lowest in the phycocyanin group (p <0.01), indicating that phycocyanin has a serum lipid-lowering effect. Furthermore, the arteriosclerosis index was also significantly improved in the phycocyanin group (p <0.0
01), it was estimated that it has an arteriosclerosis-preventing effect.

In the comparative example using soybean protein (SBP), although the mixed amount of the test sample in the feed was larger than that of the PC (phycocyanin) group, the serum total cholesterol lowering rate was higher than that of the PC group as compared with the example. The HDL-c was low in the PC group, whereas it was low in the SBP group. As a result, the arteriosclerosis index showed a significant increase (p <0.001) in the PC group, but only showed an increasing tendency in the SBP group.
No significant difference was found. From these results, it is clear that phycocyanin (PC) has a larger effect of suppressing elevation of serum cholesterol than soybean protein (SBP) and is more effective in preventing arteriosclerosis.

[0037]

EFFECTS OF THE INVENTION The present invention provides a natural lipid-derived serum lipid-lowering agent having excellent serum lipid-improving activity and few side effects, and a health food.

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B018 MD20 MD67 ME14 MF01                 4C084 AA02 BA44 CA07 DC50 MA17                       MA22 MA23 MA35 MA37 MA41                       MA43 MA52 MA66 NA06 NA07                       ZC332                 4C088 AA12 AC15 BA09 BA16 CA23                       NA06 NA07 ZC33

Claims (5)

[Claims] 1. A serum lipid-lowering agent comprising a phycobiliprotein extracted from algae as an active ingredient. 2. The serum lipid lowering agent according to claim 1, wherein the alga is a cyanobacteria of the genus Spirulina. 3. The serum lipid lowering agent according to claim 1, wherein the phycobiliprotein is phycocyanin. 4. A serum lipid lowering agent containing phycocyanin as an active ingredient. 5. A health food containing phycocyanin as an active ingredient.