KR20120051458A - Method of preparing a composition including astaxanthin and dha- and/or epa-conjugated phosphatidylserine using krill-derived lecithin, and a composition prepared by the method - Google Patents

Abstract

PURPOSE: A method for manufacturing a composition containing DHA- and/or EPA-conjugated phosphatidyl serine using krill phospholipid is provided to improve brain function and to treat vascular dementia. CONSTITUTION: A method for manufacturing a composition containing astaxanthin and DHA(docodahexaenoic acid)- and EPA(eicosapantaenoic acid)-conjugated phophatidylserine comprises a step of treating phopholipase D and serine to krill-derived lecithin. The composition contains two or more kinds selected from DHA-conjugated phophatidyl serine, EPA-conjugated phosphatidyl serine, and DHA- and EPA-conjugated phosphatidyl serine. A composition for improving brain function and cognition function contains astaxanthin and phosphatidyl serine.

Description

METHOD OF PREPARING A COMPOSITION INCLUDING ASTAXANTHIN AND DHA- AND / OR A method for preparing a composition containing astaxanthin and DHA- and / or EPA-linked phosphatidylserine using krill phospholipids EPA-CONJUGATED PHOSPHATIDYLSERINE USING KRILL-DERIVED LECITHIN, AND A COMPOSITION PREPARED BY THE METHOD}

The present invention uses at least one of DHA (docodahexaenoic acid) and EPA (eicosapantaenoic acid) using krill-derived lecithin containing natural astaxanthin. It relates to a method for preparing a composition containing phosphatidylserine in combination with, and to a composition prepared by the method.

Phosphatidylserine is a kind of fat that exists in nature, and is a substance in which a serine group (hydrophilic part), a phosphate group, glycerol and two fatty acid groups (fractional part) are combined.

The human brain accounts for about half of the dry weight of lecithin, most of which form nerve cell membranes. Phosphatidylserine is a kind of lecithin derivative, which is an important component of cell membranes, and is especially present in the brain, and it transmits information such as energy entry, release of neurotransmitters, and synaptic activity for life support activities in neuronal membranes. It is reported that he is deeply involved in the expression of nerve cells. For this reason, phosphatidylserine is called the `` brain nutrient. ''

Phosphatidylserine has been studied by numerous researchers since it was isolated by Folch in 1948. By 1997, 34 clinical trials (17 of which were blinded) were conducted, and as a result, they have been known to have functions to improve dementia, including Alzheimer's disease, and rejuvenate brain function. Many studies have been reported on the effects of improving brain function, and the effects on epilepsy, resistance to stress, and recovery of hormonal secretion rhythms have been revealed.

Conventionally, phosphatidylserine extracted from bovine cerebrum has been reported to act to increase glucose concentration in the brain, and since then, phosphatidylserine has been reported to improve dementia and memory. However, when phosphatidylserine was extracted from a cow's brain, only 1g of phosphatidylserine was available from a single cow's brain, and phospatidylserine derived from a cerebellum was developed in the UK and became a major problem worldwide. There is concern.

Recently, it has been confirmed that phosphatidylserine obtained by using a phosphatidyl group transfer reaction by an enzyme from lecithin derived from soybean has an effect of increasing glucose concentration in the brain and restoring memory impairment. Soybean-derived phosphatidylserine is now commercially available.

However, phosphatidylserine derived from soy lecithin has a different fatty acid composition from phosphatidylserine derived from cerebellum. Indeed, phosphatidylserine, which is present in animal brains, contains about 10% of DHA (docosahexaenoic acid) and has a bound structure.

DHA is known to play a very important role in brain function. DHA has been reported to play an important role in the production of phosphatidylserine in the brain in experiments with omega-3 deficient animals and is an important nutrient in the brain.

In the case of eicosapentaenoic acid (EPA), it is widely used in blood circulation improving agents such as thrombolytic dissolution and is known to be helpful for vascular dementia.

These DHA and EPA are reported to be efficiently delivered to the brain when present in phospholipid form rather than triglyceride form (Am. J. Clin. Nutr. 1998: 67, 97-103).

Thus, DHA-conjugated phosphatidylserine may be capable of delivering DHA into the brain compared to soy lecithin-derived phosphatidylserine that does not contain DHA.

Japanese Patent No. 3791951 discloses a method for producing a phosphatidylserine-containing composition having highly unsaturated fatty acids such as DHA and EPA. In this patent, as a natural lecithin raw material, (i) lecithin extracted from marine tofu tissues or (ii) fish oil or vegetable oil containing polyunsaturated fatty acids are ingested. Lecithin extracted from chicken egg yolk lecithin is used.

However, lecithin obtained from fish has not yet been mass produced or sold commercially. In addition, due to the significant decrease in oxidative stability, rancidity easily occurs, and it is difficult to obtain phospholipids having polyunsaturated fatty acids such as DHA and EPA, and all of them are disposed of. The yolk-derived lecithin contains a large amount of cholesterol, which causes arteriosclerosis, and thus, phosphatidylserine prepared by using the yolk contains negative cholesterol.

Accordingly, the present inventors, in order to solve the above problems, contains a natural astaxanthin having a very high antioxidant effect, and krill containing phosphatidylcholine, phosphatidylethanolamine, etc. combined with polyunsaturated fatty acids such as DHA, EPA Attention was paid to derived phospholipids (Krill-derived lecithin). Phospholipids with DHA or EPA may be less oxidatively stable than otherwise. Krill-derived phospholipids contain astaxanthin, which has high antioxidant potential, which can enhance oxidative stability. When such a krill-derived phospholipid is used as a raw material and treated with serine and phospholipase D, a phosphatidylserine-containing composition having a DHA content similar to that of phosphatidylserine present in the brain can be prepared.

Accordingly, an object of the present invention relates to a method for preparing a composition containing phosphatidylserine to which astaxanthin and DHA and / or EPA are bound using a krill derived phospholipid containing astaxanthin.

Another object of the present invention relates to a composition for improving brain function and cognitive function prepared by the above method and containing astaxanthin and DHA- and / or EPA-linked phosphatidylserine.

Another object of the present invention relates to a dietary supplement and pharmaceutical composition for improving brain and cognitive function, including the composition.

In accordance with the above object, the present invention is treated with phospholipid D and serine to krill-derived lecithin containing astaxanthin and bound to at least one selected from DHA and EPA. Provided is a method of preparing a composition containing phosphatidylserine bound to at least one selected from astaxanthin and DHA and EPA, comprising the step of reacting.

In accordance with another object, the present invention provides a composition for improving brain function and cognitive function, which is prepared by the above method and contains phosphatidylserine bound to at least one selected from astaxanthin and DHA and EPA.

According to another object, the present invention provides a dietary supplement and pharmaceutical composition for improving brain function and cognitive function, including the composition.

The manufacturing method of the present invention, while using krill phospholipid containing phosphatidylserine in the form of 10% or more DHA bound in a similar amount to that contained in the phosphatidylserine existing in the cerebellum, which has been proven numerous clinical results At the same time, it is very efficient in that it contains a natural astaxanthin that prevents the deterioration of oxidative stability and can easily provide a very stable composition without additional antioxidant treatment. Thus, a composition containing astaxanthin and phosphatidylserine combined with DHA and / or EPA, prepared according to this preparation method, has a similar content of DHA as that of cerebellar-derived phosphatidylserine, which has been reported to be effective in many existing clinical trials. Not only does it have excellent brain function improvement effect compared to soybean-derived phosphatidylserine that does not contain DHA, and its natural astaxanthin does not require additional antioxidants, and its storage stability is excellent due to its high antioxidant effect. Brain cell protection also has an excellent advantage.

1 is a graph showing the results of the oxidation stability test at room temperature (25 ℃) of the composition of Example 1 of the present invention.
FIG. 2 shows the results of acquisitions obtained by measuring the time to reach the escape zone within 180 seconds for 6 days of underwater maze test for the soybean-derived phosphatidylserine-administered group and the krill-derived phosphatidylserine-administered group.
Figure 3 shows the results of the phage test to remove the shelters on the seventh day of the last day of the underwater maze test for the soybean-derived phosphatidylserine-treated group and the krill-derived phosphatidylserine-treated group and to stay in the shelters area.

A method for preparing a composition containing astaxanthin according to the present invention and containing phosphatidylserine bound to DHA and / or EPA is a phospholipid derived from krill containing astaxanthin and bound to DHA and / or EPA ( Treatment with phospholipase D and serine to krill-derived lecithin.

"Krill" is a marine invertebrate that resembles a shrimp belonging to the order Euphausiacea .

Krill-derived phospholipids can be obtained by methods known in the art, for example, by pulverizing krill, followed by extraction and concentration using a solvent such as hexane, alcohol, or the like. In particular, in order to increase the content of phosphatidylcholine in krill-derived phospholipids can be extracted with alcohol only and then concentrated. Specifically, the dried krill may be pulverized and extracted under nitrogen filling for 1 hour at 20 ° C. using 95% alcohol, and then the extract is left standing for 12 hours in a refrigerated state, and the supernatant is taken under reduced pressure, concentrated and dried to obtain krill phospholipid.

The krill-derived phospholipids thus obtained include mostly astaxanthin and phosphatidylcholine combined with DHA and / or EPA, and astaxanthin may be included in an amount of 0.02 to 0.08% by weight based on the total weight of the phospholipids. DHA and EPA may be included in amounts of 10-20 wt% and 25-35 wt%, respectively, based on the total weight of fatty acids contained in phosphatidylcholine in krill phospholipids.

Phospholipase D based on the krill-derived phospholipid may be used in a 0.05 to 0.2 weight ratio, preferably 0.1 to 0.15 weight ratio, and serine may be used in a 0.5 to 3 weight ratio, preferably 1 to 2 weight ratio.

The enzyme reaction may be carried out by a method known in the art, for example, may be carried out at 35 to 45 ℃ using a solvent such as ethyl acetate for 20 to 24 hours.

Phospholipase D may be derived from microorganisms, from plants such as cabbage, and the like.

The final composition obtained may be in powder form, paste form or liquid form.

Astaxanthin is a kind of natural carotenoid pigments distributed in krill, shrimp, crab, salmon, etc., and is widely distributed in crustaceans and aquatic animals. It is red in color and is often present as a pigment protein combined with protein as a fat-soluble pigment. When crab is boiled, it becomes red because the pigment breaks down and the color of astaxanthin appears. Astaxanthin's antioxidant capacity is known to be about 100 times that of vitamin E. These antioxidant effects of astaxanthin are combined with DHA and EPA to prevent oxidative stability of phospholipids, which are vulnerable to oxidative stability, and are also positive for brain cell protection.

In addition, krill-derived phospholipids include phospholipids such as phosphatidylcholine, in which DHA is important for brain function, bound to about 10%. Enzymatic reaction of phospholipase D and serine by the method of the present invention improves cognitive ability and early dementia. Phosphatidylserine-containing compositions having a level of 10% or more of DHA bound to a level similar to that of cerebellum-derived phosphatidylserine have been reported. In addition, it is also very efficient to smoothly bleed the DHA to include a phosphatidylserine combined with EPA, such as effective for vascular diseases, so that it can be easily obtained with a high yield in a high yield.

The present invention also provides a composition for improving brain function and cognitive function, prepared by the above-described manufacturing method, containing natural astaxanthin and containing DHA and / or EPA-bound phosphatidylserine.

Preferably, the content of astaxanthin in the composition is 0.02 to 0.08% by weight, based on the total weight of the composition. The content of phosphatidylserine in the composition may be 20 to 90% by weight, in particular 20 to 40% by weight, based on the total weight of the composition. The content of DHA bound to phosphatidylserine is 10 to 20% by weight based on the total weight of fatty acids bound to phosphatidylserine, and the content of EPA may be 25 to 35% by weight.

The composition of the present invention exhibits an effect of improving brain function and cognitive function, and is used for the prevention and treatment of memory loss, memory impairment, in particular memory impairment associated with deficiency of acquisition and / or loss of activity memory, dementia, forgetfulness, Alzheimer's disease and the like. Can be used effectively.

In addition, the composition of the present invention can be used not only for the purpose of preventing and treating brain dysfunction, but also for the purpose of further improving brain function such as learning ability, memory or reflex reaction ability of a healthy person.

The compositions of the present invention may be formulated by methods known in the pharmaceutical or food arts, and may be used alone or in admixture with pharmaceutically acceptable carriers, excipients, etc. For example, it may be formulated as a liquid, syrup, capsule, and the like, which may be administered orally or parenterally.

Liquids, capsules and the like containing the composition of the present invention can be used as a dietary supplement. As used herein, the term "health supplement" refers to a health food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having useful functions for the human body or It refers to functional foods such as drinks, yogurt and cheese.

The composition of the present invention is appropriately selected according to the absorption of the active ingredient in the body, the rate of excretion, the age and weight of the patient, the sex and condition, the severity of the disease to be treated, etc., generally in adults 100 ~ 400 mg / 60kg, It is preferable to administer at 200-300 mg / 60 kg. The unit dosage form so formulated may be administered several times at regular time intervals as needed.

The composition of the present invention did not cause rancidity for 13 months in the oxidative stability test at room temperature (see Test Example 1), and in the labyrinthal learning test using aging rats compared to soybean-derived phosphatidylserine, the effect of improving spatial memory Very good (see Test Example 2). Therefore, the composition prepared in the present invention can be effectively used as a composition for improving brain function.

Hereinafter, the present invention will be described in detail with reference to examples. However, this is merely to aid the understanding of the present invention, and the scope of the present invention is not limited to the following examples.

≪ Example 1 >

200 g of L-serine was added to 400 ml of sodium acetate buffer (pH 5.6), and actinomycetes-derived phospholipase D 2g (4000 unit / g) was added, followed by stirring at 40 ° C to completely dissolve serine. Here, a solution of 100 g of krill phospholipid [DS-Krill PC30, Doosan Co., Ltd.] containing 340 ppm of astaxanthin and 35% by weight of phosphatidylcholine was mixed in 500 ml of ethyl acetate, and the mixture was stirred at 200 rpm at 24 ° C for 24 hours. The reaction was carried out for hours.

After the reaction solution was left to stand, the upper layer was collected, washed three times with water to remove residual serine and enzymes, and then the solvent layer was concentrated to contain a composition containing astaxanthin and phosphatidylserine (85.2 g, phospholipid yield: 85.2%). Obtained. The contents of phosphatidylserine and astaxanthin in the obtained composition were 30.5 wt% and 330 ppm, respectively, by HPLC analysis.

<Example 2>

200 g of L-serine was added to 400 ml of sodium acetate buffer (pH 5.6), and actinomycetes-derived phospholipase D 2g (4000 unit / g) was added, and stirred at 40 ° C to completely dissolve serine. Here, a solution of 100 g of krill phospholipid [DS-Krill PC50, Doosan Co., Ltd.] containing 450 ppm of astaxanthin and 55% by weight of phosphatidylcholine was mixed in 500 ml of ethyl acetate, and then stirred at 200 rpm at 40 ° C. The reaction was carried out for 24 hours.

After the reaction solution was left to stand, the solvent layer was recovered, washed three times with water to remove residual serine and enzyme, and then the solvent layer was concentrated to contain astaxanthin and phosphatidylserine (85.9 g, yield relative to phospholipid: 85.9%). Obtained. The contents of phosphatidylserine and astaxanthin in the obtained composition were 50.1% by weight and 448 ppm, respectively, by HPLC analysis.

<Example 3>

500 g of ethyl acetate was added to 50 g of phospholipid obtained by washing 150 g of krill phospholipid [DS-Krill PC30, Doosan Co., Ltd.] with 340 ppm astaxanthin and 35 wt% of phosphatidylcholine and removing neutral lipids. Was added to dissolve.

200 g of L-serine and 400 ml of sodium acetate buffer (pH 5.6) were added thereto, and actinomycetes-derived phospholipase D 2g (4000 unit / g) was added thereto, followed by stirring at 40 ° C. at 200 rpm and 24 ° C. at 24 ° C. The reaction was carried out for a time.

The reaction solution was left to stand, and then the upper portion was collected and washed three times with water to remove residual serine and enzyme. Then, the solvent layer was concentrated, filtered by addition of acetone, and dried under reduced pressure to obtain a composition containing astaxanthin and phosphatidylserine in powder form (35.1 g, yield: 70.2%). The contents of phosphatidylserine and astaxanthin in the obtained powder composition were 90.5% by weight and 205 ppm, respectively, by HPLC analysis.

The DHA and EPA contents in the compositions obtained in Examples 1 and 3 are shown in Table 1 below.

Composition of Example 1
(Liquid = Krill PS + krill oil) Composition of Example 3
(Krill PS Powder) EPA content 15.4 wt% 32.0 wt% DHA content 7.0 wt% 14.4 wt%

< Test Example  1> Oxidation Stability Test

Take 3 g of the composition containing astaxanthin and phosphatidylserine prepared in Example 1, and obtain the induction period with time at 85, 90, 100 ° C. using an oxidative stability measuring instrument (Rancimat), and use the Arrhenius equation. The induction period at room temperature (25 ℃) was calculated by calculating the correlation between induction period and temperature.

The result is shown in FIG . Looking at Figure 1 , rancidity did not occur after 13 months at room temperature. It is believed that the shelf life of the composition of the present invention is possible for more than one year at room temperature, it is a fairly stable composition.

<Test Example 2> Underwater Maze Test (Acquisition + Holding)

Soy phosphatidylserine (DS-PS 60, hereinafter simply referred to as soy PS) and the composition obtained in Example 3 (hereinafter referred to simply as krill PS) were aged 12 months old Sprague-Dawley (SD) rats (weight: 500). ~ 530g, 6 animals per dose group) 50mg / kg and 20mg / kg daily for a week and the maze learning test was conducted.

Specifically, in the soybean PS-administered group and the krill PS-administered group, acquisition results obtained by measuring the time to reach the shedding zone within 180 seconds for 6 days are shown in FIG. 2 . In Fig. 2 , NORMAL represents a normal group, AG represents a control group (aging rats) ingested nothing, AG-SOY-PS50 represents a soybean PS administration group administered 50 mg / kg soybean PS daily to aging rats, AG-KRILL-PS20 represents a krill PS-administered group in which 20 mg / kg of krill PS of Example 3 was administered daily to aging rats. Referring to FIG. 2 , the acquisition trial measuring the time to reach the escape within 180 seconds appeared similarly without significance on the first day, but the time required to reach the escape on the last six days as learning progressed. Significant differences were shown between groups. As a result of the post hoc test according to the measurement days, the time to reach the escape zone was statistically significantly reduced in the soybean PS-administered group and the Krill PS-administered group of Example 3, and showed an enhancement effect on the performance of spatial cognitive memory learning. In particular, the krill PS group received a relatively short time to reach the shelter despite ingesting a smaller amount than the soybean PS group, which means that krill PS of the present invention is more effective in improving brain function and cognitive function than conventional soybean PS. It can be seen that it is effective.

On the other hand, gripping was performed to measure the degree of staying in the area of the escape zone by removing the escape zone on the seventh day, which is the last day of the underwater maze study. The results are shown in Figure 3. After removing the shelters, the rats with good memory will have a longer stay in the section where the shelters were located. In the case of Krill PS, the retention time was longer in the section where the shelter was located than soybean PS, which indicates that krill PS of the present invention has better spatial memory than soybean PS.

The compositions of the present invention can be used to prepare a pharmaceutical formulation or food as follows.

Manufacturing example  1: Preparation of Capsule

200 mg of the composition of Example 1

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Manufacturing example  2: Preparation of Capsule

100 mg of the composition of Example 2

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Manufacturing example  3: Liquid  Produce

200 mg of the composition of Example 2

20 g of sugar

20 g of isomerized sugar

Lemon incense quantity

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml brown bottles and sterilized to prepare a liquid.

Manufacturing example  4: manufacture of beverage

5 wt% of the composition of Example 1, 0.05 wt% of food coloring, 0.05 wt% of orange essence, 5.0 wt% of fructose, 0.1 wt% of citric acid, 0.05 wt% of vitamin C was added to prepare a composition containing a general functional beverage base Next, a suitable amount of purified water was added to prepare a beverage.

Claims (3)

Phospholipase D and serine were treated by krill-derived lecithin (Krill-derived lecithin) containing astaxanthin and combined with at least one selected from docodahexaenoic acid (DHA) and eicosapantaenoic acid (EPA). A method for preparing a composition containing phosphatidylserine, which is combined with at least one selected from astaxanthin, DHA and EPA, comprising reacting the same. The method of claim 1,
Wherein said composition comprises two or more selected from DHA-linked phosphatidylserine, EPA-linked phosphatidylserine, and DHA- and EPA-linked phosphatidylserine. A composition for improving brain function and cognitive function prepared by the method according to claim 1 or 2 and containing astaxanthin and phosphatidylserine.

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