JP2003012520A - Antioxidant and food and drink containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antioxidant having suppressing action for peroxidizing lipids of biological tissue by oral administration, and to provide foods and drinks containing the antioxidant. SOLUTION: This antioxidant having suppressing action for peroxidizing lipids of biological tissue is obtained by including plasmalogen as the active ingredient. The antioxidant preferably contains >=1 mass % plasmalogen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antioxidant having an action of suppressing lipid peroxidation of living tissues by oral administration, and foods and drinks containing the same.

[0002]

2. Description of the Related Art 1-Alkenyl-2-acylglycerophospholipids (plasmalogens), which are constituents of biological membranes
Is a unique phospholipid having a vinyl ether bond at the 1-position of the glycerol skeleton and is abundantly contained in mammalian heart, brain, skeletal muscle, erythrocyte, and gill of crustaceans.

Since all of these tissues have a high oxygen consumption amount, a relationship with oxygen toxicity in the living body has been pointed out. One of the reasons that plasmalogens are unevenly distributed in tissues with high oxygen consumption is their role as antioxidant lipids in biological membranes. In recent years, liposomes (Zommara et a
l .: Free Radicals Biol. Med., 18, 599-602, 199
5), low-density lipoprotein (Hahnel et al .: Bioche
m. J., 340, 377-383, 1999), cultured cells (Zoeller et.
al .: J. Biol. Chem., 263, 11590-11596, 1988) has revealed the antioxidative effect of plasmalogens by in vitro studies.

[0004]

[Problems to be Solved by the Invention] Beef, pork, chicken,
Plasmalogens are contained in meat and seafood (Fogerty et al .: Int. J. Food Sci. Technol., 2
6,363-371, 1991), we ingest plasmalogens through our daily diet. However, there are few reports on the effects of oral ingestion of plasmalogen on the oxidation of living body. For example, ischemic heart disease, coronary arteriosclerosis, and Alzheimer's disease have been reported as pathological conditions related to the oxidation of biological membrane components. Plasmalogen is significantly decreased in platelets in ischemic heart disease, serum in patients with coronary arteriosclerosis, and brain in patients with Alzheimer's disease (Matsuo et al .: Jikei University, 103, 751-759, 1).
988. Suzuki et al .: Atherosclerosis, 14, 155-162, 1986. ; Gin
sberg et al .: J. Neurochem., 70, 2533-2538, 199
8) indicates that the abnormal metabolism of phospholipids may be involved in these diseases. That is, if it is possible to enhance the antioxidant capacity of the living body by ingesting plasmalogen, it is considered to be very important from the viewpoint of improving and preventing these diseases.

Therefore, it is an object of the present invention to provide an antioxidant having an action of suppressing the peroxidation of lipids of living tissues by oral administration, and foods and drinks containing the same.

[0006]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that by ingesting rat plasmalogen orally, plasma components and organs (liver, heart, brain) It has been found that lipid peroxidation can be suppressed, and the present invention has been completed based on this fact.

That is, one aspect of the present invention is an antioxidant characterized by containing a phospholipid represented by the following general formula (I) as an active ingredient.

[0008]

[Chemical 2]

The above antioxidant contains 0.1% of the above phospholipid.
It is preferable that the content is at least mass%. According to the above-mentioned invention, it is possible to provide an antioxidant having an action of suppressing lipid peroxidation of in vivo tissues by oral administration. By ingesting this antioxidant, it is possible to effectively suppress the peroxidation of lipids in the tissues of the living body, and it can be expected to have an improving / preventing effect on ischemic heart disease, coronary arteriosclerosis, Alzheimer's disease and the like.

Another aspect of the present invention is a food or drink containing the above antioxidant. The food or drink preferably contains the antioxidant in an amount of 0.01% by mass or more in terms of the phospholipid. According to the above invention, it is possible to provide a food or drink to which the physiologically active effect of the above phospholipid is added.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The active ingredient of the antioxidant of the present invention is
A phospholipid represented by the following general formula (I) (hereinafter, simply referred to as plasmalogen).

[0012]

[Chemical 3]

R ³ in the above general formula (I) is preferably an alkyl group having 1 to 20 carbon atoms, and specific examples thereof include a dodecyl group, a tetradecyl group, a hexadecyl group, an octadecyl group and an icosanyl group. Also, R ⁴ is
Examples of the alkyl group include those similar to R ² above,
The highly unsaturated fatty acid residue, specifically, octadecadienoyl group, octadecatrienoyl group, icosatetraenoyl group, icosapentaenoyl group, docosatetraenoyl group, docosapentaenoyl group, Examples thereof include docosahexaenoyl group.

The antioxidant of the present invention preferably contains 0.1% by mass or more of the above plasmalogen, and 10% by mass.
It is more preferable to include the above.

The above-mentioned plasmalogen is contained in meat such as beef, pork and chicken, seafood, etc., and total lipids are extracted from these natural products, and the phospholipid fraction containing plasmalogen is extracted from this total lipid fraction. Can be obtained by further separating it and further purifying it if necessary. In the present invention, it is preferable to extract and separate from natural products having a high plasmalogen content, for example, the heart and brain of mammals such as cows and pigs, and marine invertebrates such as cephalopods, shellfish and crustaceans.

Examples of the method for extracting total lipids from the above natural products include, for example, the Folch method (Folch et al .: J. Biol. Che.
m., 226, 497-505, 1957), Bligh & Dyer method (Bligh et
al .: Can. J. Biochem. Physiol., 37, 911-917, 195
9) etc.

Further, as a method for separating the phospholipid fraction containing plasmalogen from the above total lipid fraction, for example, an acetone precipitation method (supervised by Tamio Yamakawa: Biochemistry Experimental Course 3, Lipid Chemistry (edited by the Japanese Biochemical Society), p.19-20, 1963, Tokyo Kagaku Dojin, Seppack column method (James et al .: Lipds,
23, 1146-1149, 1988) and the like.

Further, as a method for purifying plasmalogen from the phospholipid fraction containing plasmalogen,
For example, a method of decomposing diacyl-type phospholipid by treating the above phospholipid fraction with a weak alkali, and a method of purifying by using phospholipase A derived from Crotalus atrox (Gottfried et a
l .: J. Biol. Chem., 237, 329, 1962) and the like. Examples of the weak alkali treatment include a treatment in methanolic 0.1N KOH at 10 ° C.

Plasmalogens convert glycerin into alkenyl glyceryl ether (Pianta, for example).
dosi et al .: J. Pharm. Sci., 50, 978, 1961), and then chemically synthesized by esterification or phosphorylation. However, since this method is complicated, and the organic solvent and catalyst used are harmful to the human body, it cannot be said that the method is very suitable for industrial production, and in terms of safety and productivity. Also, a method of extracting and separating from the above natural product is preferable.

The antioxidant of the present invention comprises, as other antioxidant components, ascorbic acid, gallic acid, reduced glutathione,
Vitamin E, carotenoid, flavonoid, BHT, B
HA and the like may be included. By blending the above-mentioned antioxidant component, a synergistic effect with plasmalogen can be expected.

In addition to the above-mentioned basic components, the antioxidant of the present invention can appropriately contain excipients, sugars, fragrances, dyes, emulsifiers, preservatives and the like.

The product form of the antioxidant of the present invention is not particularly limited and can be appropriately selected according to the use form, for example, tablets,
Powders, granules, capsules, pastes, emulsions, solutions and the like can be mentioned.

The effective daily intake of the antioxidant of the present invention is 30 to 1000 mg / kg body weight, more preferably 300 to 1000 mg / kg body weight in terms of plasmalogen.
Is.

The antioxidant of the present invention may be added to foods and drinks such as soft drinks, tablet confectionery, snacks, powdered soups and sausages. In that case, the addition amount per serving is preferably 30 to 1000 mg in terms of plasmalogen,
-1000 mg is more preferable. If the amount of antioxidant added is less than 30 mg in terms of plasmalogen, sufficient antioxidant effect cannot be expected even if ingested, and if it exceeds 1000 mg, excessive intake of highly unsaturated fatty acids that bind to plasmalogen On the contrary, there is a risk of increasing the oxidizability of the living body, which is not preferable.

[0025]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 Japanese starfish (Astropecten scopariu), which is a kind of starfish
s Valenciennes) (from Ishikari Bay and Esashi Coast) was used as a raw material to prepare plasmalogens.

Total lipid extraction was performed by the Folch method (Folch et al .: J.
Biol. Chem., 226, 497-505, 1957), and
Acetone precipitation method for preparation of phospholipids (supervised by Tamio Yamakawa: Laboratory for biochemistry 3, Chemistry of lipids (edited by the Japanese Biochemical Society), p.19-2
0, 1963, Tokyo Kagaku Dojin).

Specifically, the mussels were ground in a frozen state, about 4 times its volume of acetone was added, the mixture was allowed to stand at 4 ° C. for one day and night, and suction-filtered to obtain an acetone-soluble fraction (non-phospholipid). It was removed and the residue was collected. About 4 of this residue
A double volume of acetone was added and the same operation was repeated.

Approximately 4 times its volume of chloroform / methanol solution (chloroform: methanol =
2: 1, v / v) was added, and the mixture was sufficiently stirred, allowed to stand for a day and night, and then a chloroform / methanol-soluble fraction was collected by suction filtration. Water was added to this chloroform / methanol-soluble fraction to divide it into two layers, and the chloroform layer was recovered and concentrated to obtain a total lipid fraction. This total lipid fraction is 10
After adding dropwise to a double volume of cold acetone while stirring, the mixture was allowed to stand at 4 ° C. for one day and then the supernatant was removed to obtain a precipitate (crude phospholipid). This operation was repeated twice more to prepare a starfish-derived phospholipid fraction.

The obtained phospholipid fraction was analyzed by the method of Dawson et al. (Dawson et al .: Biochem. J., 75, 45-53, 1960) to find that the diacyl phospholipid was 47.6% by mass and plasma. It contained 35.9% by mass of a logen and 16.5% by mass of an alkylacyl phospholipid.

Example 2 A scallop-derived phospholipid fraction was prepared in the same manner as in Example 1 using frozen scallop. When this phospholipid fraction was analyzed by the same method as in Example 1, it contained 25.5% by mass of plasmalogen.

Further, in order to enhance the purity of plasmalogen in this phospholipid fraction, the diacyl phospholipid was preferentially decomposed by treatment with a weak alkali. Specifically, 100
The above phospholipid fraction (8.3 g) was added to ml of methanolic 0.1 N KOH, and the mixture was stirred at 10 ° C. It should be noted that in order to determine an appropriate decomposition reaction time (0, 20,
The reaction solution was collected at 30 and 40 minutes), washed with acetone, pulverized, and the change in the plasmalogen content in the reaction solution was measured. The result is shown in FIG.

From FIG. 1, 30 phospholipid fractions derived from scallop were obtained.
It can be seen that the plasmalogen content is increased 1.8 times (45.8% by mass) by the weak alkali treatment for a minute. Therefore, it was found that a treatment time of about 30 minutes with a weak alkali is suitable.

Example 3 Using the starfish-derived phospholipid fraction (plasmalogen content 35.9% by mass) prepared in Example 1 and the diacyl phospholipid prepared from herring testis as a comparative control, an organ by oral ingestion was used. The effect on lipid peroxidation of (liver, heart, brain) was examined.

1. Preparation of Feed The test feed was prepared according to the AIN-93G composition having a lipid content of 7% by mass. Specifically, 5% by mass of soybean oil and a diet in which the remaining 2% by mass is replaced with a diacyl-type phospholipid (hereinafter referred to as DP diet), and a diet in which the remaining 2% by mass is replaced with a starfish-derived phospholipid fraction ( Hereinafter, the PP feed) was prepared. As a result of the component analysis, D
In the P diet, most of the phospholipid components (94.4 mol%) were diacyl phospholipids, and plasmalogen and alkylacyl phospholipids were few. On the other hand, the PP feed contained 47.6 mol% of diacyl phospholipids, 35.9 mol% of plasmalogens, and 16.5 mol% of alkylacyl phospholipids, and had a composition with a higher ratio of plasmalogens than the DP group. In addition, each prepared feed was stored at -70 ° C until the start of feeding after filling the head space of the container with nitrogen gas.

2. Animal experiment 4-week-old male Wistar rats (purchased from CLEA Japan, Inc.) were divided into 7 cages under the conditions of a temperature of 22 ± 2 ° C, a humidity of 45 ± 3%, and a 12-hour light / dark cycle. After preliminarily breeding with a feed MF (trade name, manufactured by Oriental Yeast Co., Ltd.) for 12 days, rats with no abnormal growth were selected and divided into 2 groups (7 animals per group). The DP feed was 20 g per animal per day for 4 weeks. Also, water was freely available. During the test period,
No effects on the weight gain and the food intake of the rats in each group were observed.

After 12 hours of fasting on the last day of feeding for 4 weeks, the abdomen was opened under anesthesia with sodium pentobarbital, and blood was collected by cardiac puncture under EDTA.2K and centrifuged (1400).
Plasma was separated by (× G, 10 minutes), and low density lipoprotein (hereinafter referred to as LDL) was obtained by the heparin precipitation method.

Next, the liver, heart and brain were removed, perfused with phosphate buffered saline (PBS, pH 7.4, 4 ° C.) and bleeding, and the mass of each organ was measured to immediately obtain liquid nitrogen. It was frozen at and stored at -70 ° C until analysis.

3. Measurement of Lipid Peroxidation Degree of Organs The lipid peroxidation degree (TBA value) was measured by a fluorescence-HPLC method (Fukunaga et al .: J. Chromatogr., 621, 77-81, 199).
According to 3), tetraethoxypropane was used as a standard substance and quantified by the external standard method.

Specifically, 100 μl of 10% homogenate of each organ was placed in a test tube, and 1.9 ml of 0.1.
2% TBA (2M acetate buffer, pH 3.5) solution and 20
μl of 0.5% butylhydroxytoluene in ethanol was added and heated at 95 ° C. for 45 minutes. 2m after cooling
1 n-butanol was added, stirred well and centrifuged (1
200 × G, 10 minutes), upper layer (butanol layer)
Was collected, 3 times volume of acetonitrile was added thereto to prepare a sample, and HPLC analysis was performed under the following conditions.

Column: Nucleosil 100-C18 (10μ
m, 4.6φ × 250 mm) (trade name, manufactured by GL Science Co., Ltd.) ・ Solvent: acetonitrile: water = 2: 8 (v / v) ・ Flow rate: 1.0 ml / min ・ Temperature: room temperature ・ Detection: fluorescence ( EX. = 515 nm, Em. = 553n
m) The measurement results were expressed as MDA (pmol) per mg of organ protein. The results of measuring the lipid peroxidation degree of each organ are shown in FIG.

From FIG. 2, the TBA values of the liver and heart are D
It can be seen that the PP group shows a lower tendency than the P group.
In addition, the TBA value in the brain of the PP group (141.0 pmo
ImDA / mg protein is the value of DP group (2
Since the level was significantly lower than 24.0 pmol MDA / mg protein), it was revealed that ingestion of plasmalogen can suppress lipid peroxidation of each organ.

4. Examination of oxidative stability against iron-dependent lipid peroxidation reaction of LDL and each organ FeSO ₄ as a lipid peroxidation promoter was added to a homogenate prepared by adding 4 times its volume of physiological saline to the above LDL and each organ tissue. A final concentration of physiological saline containing 10μ
After adding M to obtain M, incubation was carried out at 37 ° C., 20 μl of the reaction solution was sampled at regular intervals, and the TBA value was measured according to the above fluorescence-HPLC method. The result is shown in FIG.

From FIG. 3, in the liver homogenate and the heart homogenate, the TBA value of the PP group was higher than that of the DP group.
It can be seen that the reaction always keeps a low value throughout 240 minutes. On the other hand, the TBA value of the brain homogenate was not significantly different between the two groups. Further, the LDL of the PP group also tended to be less susceptible to peroxidation than the DP group.

From the above results, it was revealed that ingestion of plasmalogen increases the oxidative stability of the liver, heart and LDL.

Example 3 Table 1 shows formulation examples of gelatin soft capsules containing the antioxidant of the present invention.

[0046]

[Table 1]

[0047]

As described above, according to the present invention, by containing plasmalogen as an active ingredient, it is possible to provide an antioxidant having an effect of suppressing lipid peroxidation of in vivo tissues by oral administration. . By ingesting this antioxidant, it is possible to effectively suppress the peroxidation of lipids in the tissues of the living body, and it can be expected to have an improving / preventing effect on ischemic heart disease, coronary arteriosclerosis, Alzheimer's disease and the like.

[Brief description of drawings]

FIG. 1 is a chart showing changes in plasmalogen content when a scallop-derived phospholipid fraction is weakly alkali-treated.

FIG. 2 is a chart showing the results of measuring the lipid peroxidation degree of each organ.

FIG. 3 is a chart showing the results of measuring the oxidative stability of LDL and each organ against iron-dependent lipid peroxidation.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C09K 15/32 C09K 15/32 D C07F 9/10 A // C07F 9/10 A23L 2/00 F (72) Inventor Kikuchi Kazuaki 5-8-13 Ogawashinmachi, Yaizu-shi, Shizuoka F-Term (reference) within Yaizu Suisan Chemical Co., Ltd. (reference) 4B017 LC03 LK10 LK17 4B018 MD16 MD17 MD45 ME06 MF01 4C086 AA01 AA02 DA34 MA01 MA04 MA52 NA14 ZC37 4H025 AA63 A67 AB83

Claims (5)

[Claims] 1. An antioxidant containing a phospholipid represented by the following general formula (I) as an active ingredient. [Chemical 1] 2. The antioxidant according to claim 1, containing 0.1% by mass or more of the phospholipid. 3. The antioxidant according to claim 1 or 2, which has an action of suppressing the peroxidation of lipids of tissues in a living body by oral administration. 4. A food or drink containing the antioxidant according to any one of claims 1 to 3. 5. The food or drink according to claim 4, which contains the antioxidant in an amount of 0.01% by mass or more in terms of the phospholipid.