KR101297339B1 - Pharmaceutical Composition and Functional Food Having Anti-Alzheimer Activity Containing Peptides from Fish Skins - Google Patents

Abstract

The present invention discloses an anti-Alzheimer's pharmaceutical composition and health functional food containing a hydrolyzate of a protein forming a fish shell and an oligopeptide which is a part thereof as an active ingredient. The peptide according to the present invention inhibits the activity of beta-secretase, which causes Alzheimer's disease, and because it is composed of di-peptides, it does not cause degradation by hydrolase in vivo even upon oral administration. It is expected to have a good physiologically active effect because it is easily absorbed and digested in vivo. In particular, since it is a component derived from a living body, there is no fear of side effects, and the safety is also good, and since it is obtained from the shells of fish that have been mostly discarded, it is possible to actively use the marine by-products.

Description

Pharmaceutical Composition and Functional Food Having Anti-Alzheimer Activity Containing Peptides from Fish Skins}

The present invention relates to an anti-alzheimer's composition, and more particularly, to an anti-alzheimer's composition containing a peptide derived from fish shell as an active ingredient.

In Korea, surrounded by oceans on three sides, it is necessary to focus research and development on the potential of functional products, such as pharmaceuticals, health foods, or cosmetics, by using substances contained in living organisms at sea. have. In particular, considering the delay in the development of new medicines or the side effects of these medicines according to the conventional chemical synthesis method, various biologically active ingredients contained in marine organisms inhabiting the sea are utilized for new antibiotics as well as anticancer drugs. In the future, much more research and development should be carried out on the potential as an active ingredient for the treatment and prevention of intractable diseases such as diabetes treatment.

By the way, there is a lot of by-products that do not need after processing aquatic products, including aquatic product processing plants, but most of the by-products are currently disposed of because there is little utilization measures for this. These by-products are mainly shells, shells, endothelium, intestines, fish bones, etc. of marine products. Most of these by-products are used or disposed of as food, so they cannot be used efficiently, causing environmental pollution. However, these by-products contain a large amount of useful bioactive substances such as proteins, carbohydrates, and fats, and there is a search for ways to utilize these substances.

Accordingly, in recent years, many studies have been made to apply to various industrial fields using protein hydrolysates derived from marine organisms. In particular, research results have been reported that the protein hydrolysates derived from marine organisms have various pharmacological effects such as antioxidant, antihypertensive, anticancer and anti-inflammatory. Value is emerging.

The skates (Skate, Raja kenojei ), a flat- bodied cartilaginous fish belonging to the smelt and stingray family, are found all over the world from shallow to over 2,700 meters across the Arctic waters. In Korea, since Mokpo has been widely used as traditional fermented foods such as skate sashimi, research on nutritional analysis of skated fermented foods has been mainly conducted, and all internal organs and skins that are not used as fermented foods are discarded. It was a situation. However, since it is known that skates and skates fermented foods contain a large amount of bioactive ingredients such as anserine, taurine, and alanine / lysine, research or patents to obtain bioactive ingredients from skates Is being reported.

For example, Korean Patent No. 607371 (Patent Document 1) discloses that the hot water extracts of the edible part, viscera, cartilage, and brain parts of skates have anti-cancer effects, and the hot water extracts of the internal organs and edible parts of skates are ACE (angiotensin). It has also been reported that there is antihypertensive activity such as inhibiting the activity of Converting Enzyme (Lim, HS 2003. ACE Inhibitory Materials from Raja kenojei.Korean J. Life Sci. , 13, 668-674, Non-Patent Document 1 ; Korean Patent Publication No. 2005-93477, Patent Document 2)). In addition, the study showed that kenojeinin I, an antimicrobial peptide isolated from matured skate skin, has the effect of controlling the growth of microorganisms such as Bacillus subtilis and E. coli (Cho, SH et al. 2005. Kenojeinin I, antimicrobial peptide isolated from the skin of the fermented skate, Raja kenojei.Food Hydrocolloids , 26, 581-587, Non-Patent Literature 2), the liver oil extracted from skates reduced LDL-cholesterol, increased HDL-cholesterol, and decreased triglycerides to reduce coronary artery disease It contains significant amounts of n-3 fatty acids, such as EPA and DHA, which are effective in preventing (NAM, HK, Lee, MK 1995. Studies on the fatty acids and cholesterol level of Raja Skate. J. Korean Oil Chem. Soc. , 12, 55-58, nonpatent literature 3).

On the other hand, salmon (salmon, Oncorhynchus keta ) is a saltwater fish with salmon- headed salmon, which are traced back to the river for spawning in the fall. Young salmon return to the sea a few weeks after hatching in spring, mature three to four years later, and return to their original streams. In Korea, it is found in some streams that flow into the East and South Seas, and is distributed in Japan, Russia, and North America. In Japan, salmon is used for foods such as salted products, salted fish, smoked products, and dried products.

Recently, however, some patents have been published on the physiological activity of salmon. Protamine, a nucleoprotein contained in salmon sperm, has antimicrobial activity against gram-positive bacteria such as Bacillus subtilis , Staphylococcus aureus , Enterobacter aerogenes , gram-negative bacteria such as Pseudomonas aeruginosa , and E. coli, and yeasts such as Candia utilis. (Joo. DS et al. 2000. Antimicrobial and Antioxidant Activity of Protamine Prepared from Salmon Sperm, Korean J. Food Sci. Technol. 32, 902-907, Non-Patent Document 4). In addition, the Republic of Korea Patent Registration No. 1048265 (Patent Document 3) and No. 1048271 (Patent Document 4) described that the dried flesh of salmon caught in the sea near the Republic of Korea is effective in the prevention or treatment of hyperlipidemia or arteriosclerosis. However, there is a great need to utilize salmon, which is not widely used in Korea, for medicine or food.

On the other hand, squids, cuttlefish, Todarodes pacificus belonging to the mollusks, cephalopods, Jessia, and teothoidea are the general marine molluscs and is one of the representative marine organisms consumed for food in Korea. Squid is not only used as a model animal for human diseases, but also as a cosmetic, for example, as a constituent of hair dyes and farmer compositions by utilizing the ink that the squid emits. In particular, recently, attempts have been made on the possibility of using squid as an active ingredient such as drugs.

In Korean Patent Publication No. 2008-94983 (Patent Document 5), the bone powder of squid can be used as an active ingredient of a pharmaceutical composition or health functional food for the prevention and treatment of gastrointestinal diseases such as gastric ulcer, duodenal ulcer, gastritis and stomach cramps. In Korea Patent Registration No. 1017734 (Patent Document 6) and Republic of Korea Patent Publication No. 2010-0056140 (Patent Document 7), the fraction obtained by separating the cuttle meat and fin powder using an organic solvent such as alcohol is hyperlipidemia It is disclosed that it is effective in the treatment and prevention of atherosclerosis. Recent reports suggest that angiotensin I-converting enzyme (ACE) converts peptides isolated from squid shells to angiotensin II, which has physiological effects such as angiotensin I from angiotensin I. (Lee. JK et al. 2011. Purification of Angiotensin I-Converting Enzyme Inhibitory Peptide from Squid T odarodes pacificus Skin, Kor J. Fish Aquat. Sci. 32, 118-125, Non-Patent Document 5)

However, skates, squid and salmon are still commonly used for food, such as fermented products and dried products. Therefore, even though it is valuable to use, such as skates, squid shells and salmon shells that can not be used for food contains a large amount of protein, the situation is mostly discarded as a by-product of fish processing. Accordingly, it is necessary to study the functions of the bioactive substances included in the fish processing by-products such as skate shells, squid shells and salmon shells, and find ways to be utilized in other industrial fields.

1. Republic of Korea Patent No. 607371 2. Korean Patent Publication No. 2005-93477 3. Republic of Korea Patent No. 1048265 4. Republic of Korea Patent No. 1048271 5. Korean Patent Publication No. 2008-94983 6. Republic of Korea Patent No. 1017734 7. Republic of Korea Patent Publication No. 2010-0056140

Lim, H.S. 2003.ACE Inhibitory Materials from Raja kenojei. Korean J. Life Sci., 13, 668-674 2. Cho, S. H. et al. 2005. Kenojeinin Ⅰ, antimicrobial peptide isolated from the skin of the fermented skate, Raja kenojei. Food Hydrocolloids, 26, 581-587 3.NAM, H.K., Lee, M.K. 1995. Studies on the fatty acids and cholesterol level of Raja Skate. J. Korean Oil Chem. Soc., 12, 55-58 4. Joo. D.S. et al. 2000. Antimicrobial and Antioxidant Activity of Protamine Prepared from Salmon Sperm, Korean J. Food Sci. Technol. 32, 902-907 5. Lee. J.K. et al. 2011.Purification of Angiotensin I-Converting Enzyme Inhibitory Peptide from Squid Todarodes pacificus Skin, Kor J. Fish Aquat. Sci. 32, 118-125

The present invention has been proposed to solve the above problems, an object of the present invention is to provide an anti-Alzheimer's pharmaceutical composition containing a peptide derived from the protein contained in the shell of the fish as an active ingredient.

Another object of the present invention is to provide a method for utilizing a protein-derived peptide contained in a fish shell that is mostly disposed of in a food engineering composition, for example, a dietary supplement.

Other advantages and objects of the present invention will become more apparent from the following detailed description of the invention and the accompanying drawings.

The present invention relates to a pharmacological composition and health functional food having an anti-Alzheimer's effect containing as an active ingredient a peptide derived from fish shells that are currently mostly disposed of.

According to an aspect of the present invention, there is provided a pharmaceutical composition having a prophylactic or therapeutic effect of Alzheimer's disease, which comprises the peptide of SEQ ID NO: 21 as an active ingredient in SEQ ID NO: 2.

The peptides of SEQ ID NO: 12 to SEQ ID NO: 16 are hydrolysates or parts of the hydrolyzate treated with papain of the protein constituting the salmon shell, and the peptides of SEQ ID NO: 17 to SEQ ID NO: 21 constitute a squid shell The protein may be hydrolyzate treated with pepsin or may be part of this hydrolyzate.

If the above-described peptide has anti-Alzheimer's activity through beta-secretase (β-secretase) inhibitory effect, preferably the peptide may be included in the pharmaceutical composition at a concentration of 0.001 ~ 10.0 mg / ㎖.

Among the aforementioned peptides, the peptide having the leucine bound to the C-terminus may be contained as an active ingredient, and particularly preferably, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15 to SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21 It contains a peptide as an active ingredient, and is selected among the peptides of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21.

In addition, according to another aspect of the present invention provides a health functional food for the prevention or improvement of Alzheimer's disease containing the peptide of SEQ ID NO: 21 in SEQ ID NO: 2 as an active ingredient.

In the present invention, the beta-secretase closely related to Alzheimer's disease, which is a degenerative brain disease, is a peptide component, which is a hydrolysis product of a protein having the highest content among body components of salmon shells, squid shells and skates, which are mostly disposed of. It was confirmed that the activity of was greatly suppressed.

Accordingly, when using a peptide, an active ingredient that inhibits the activity of beta-secretase extracted from the shells of these fishes as an active ingredient, depending on the purity of the active substance, as well as pharmaceuticals such as pharmacological composition, health It can be used as a supplement, and its value is expected to be very high when applied to the food industry and the pharmaceutical industry including health functional food.

Particularly, peptides whose physiological activity has been confirmed according to the present invention are low molecular peptides such as oligopeptides or less than 10 or less constituent amino acids or dipeptides / tripeptides / tetrapeptides, which are degraded by hydrolases in the body upon oral administration. It is expected to be easily applied as an active ingredient in medicines and health functional foods because of its excellent safety and high absorption and digestion rate.

Accordingly, it is possible to industrially apply fish shells, which are currently disposed of mostly as a by-product of fish processing, and can be used without side effects on the human body as components derived from living bodies.

1 is a graph measuring the beta-secretase inhibitory activity of a product treated with various proteolytic enzymes after grinding a salmon shell according to an embodiment of the present invention, wherein the activity of papain hydrolyzate is Shows the best
Figure 2 shows the result of column chromatography for the papain hydrolyzate of the salmon shell according to another embodiment of the present invention. (A) is a graph showing the three fraction components (primary fraction components), (B) is a graph measuring the inhibitory activity of beta-secretase of each fraction component separated by column chromatography, F1 fraction The ingredient shows the best inhibitory activity.
Figure 3 shows the results of the separation of the most excellent fractions of the beta-secretase of the primary fraction of papain hydrolyzate of salmon shells according to another embodiment of the present invention by HPLC again. (A) is a graph showing the separation of eight fractions (secondary fractions) by HPLC, (B) is a graph measuring the inhibitory activity of beta-secretase of each fraction separated by HPLC As a result, the third fraction shows the highest inhibitory activity.
4 is a result of re-separation and purification of the third best fraction by HPLC analysis, (A) is a graph showing that one fraction component was obtained, and (B) is a beta-secretase of this fraction component It is a graph measuring inhibitory activity.
Figure 5 is a graph showing the results of MS / MS analysis for the hydrolyzate fraction derived from salmon shells showing the best beta-secretase inhibitory activity in HPLC analysis.
Figure 6 is a graph measuring the beta-secretase inhibitory activity of the product treated with various proteolytic enzymes after grinding the cuttlefish shell according to an embodiment of the present invention, the activity of the pepsin (Pepsin) hydrolyzate Shows the best
Figure 7 shows the result of column chromatography for the pepsin hydrolyzate of the cuttlefish shell according to another embodiment of the present invention. (A) is a graph showing six fraction components (primary fraction components), (B) is a graph measuring the inhibitory activity of beta-secretase of each fraction component separated by column chromatography, F3 fraction The ingredient shows the best inhibitory activity.
Figure 8 shows the results of the separation of the separation component having the highest inhibitory activity of beta-secretase among the primary fraction of the pepsin hydrolyzate of the cuttlefish shell according to another embodiment of the present invention again by HPLC. (A) is a graph showing the separation of seven fractions (secondary fractions) by HPLC, (B) is a graph measuring the inhibitory activity of the beta-secretase of each fraction separated by HPLC As the fifth fraction component (E) shows that the highest inhibitory activity.
9 is the result of re-separation and purification of the fifth fraction which was the best in HPLC analysis, (A) is a graph showing that one fraction component was obtained, and (B) is a beta-secretase of this fraction component It is a graph measuring inhibitory activity.
Figure 10 is a graph showing the results of MS / MS analysis for the hydrolyzate fraction derived from the squid shell showing the best beta-secretase inhibitory activity in HPLC analysis.
FIG. 11 is a graph illustrating beta-secretase inhibitory activity of products treated with various proteolytic enzymes after grinding a red sea shell, according to an embodiment of the present invention, and the activity of Neutrase hydrolyzate. Shows that this is the best.
FIG. 12 shows the results of column chromatography separation of neutrase-treated hydrolysates from skates-derived protein according to another embodiment of the present invention. (A) is a graph showing the four fraction components (primary fraction components), (B) is a graph measuring the inhibitory activity of beta-secretase of each fraction component separated by column chromatography, F4 fraction The ingredient shows the best inhibitory activity.
FIG. 13 shows the results of HPLC separation of the fraction having the highest inhibitory activity of beta-secretase among the primary fractions of the nutrase hydrolyzate of the skate, according to another embodiment of the present invention. (A) is a graph showing separation of four fractions (secondary fractions) by HPLC, (B) is a graph measuring the inhibitory activity of beta-secretase of each fraction separated by HPLC As a result, the second fraction shows the highest inhibitory activity.
FIG. 14 is a result of re-separation and purification of the second best fraction by HPLC analysis, (A) is a graph showing that one fraction component was obtained, and (B) is a beta-secretase of this fraction component It is a graph measuring inhibitory activity.
Figure 15 is a graph showing the results of MS / MS analysis for fractions derived from the skate skin showing the best beta-secretase inhibitory activity in HPLC analysis.

The present inventors have completed the present invention by studying the bioactive effects of these components in consideration of the fact that most of them contain large amounts of protein components in shells derived from fish that are currently disposed of. The present invention is a peptide derived from fish shells, oligopeptides having approximately 10 amino acids, and particularly preferably dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides consisting of 2 to 6 amino acids in Alzheimer's disease. It was found to inhibit beta-secretase activity, which is closely related to disease.

Degenerative brain diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease and Amyotrophic lateral sclerosis (ALS) are incurable diseases that modern medicine has not solved. Among them, Alzheimer's disease is a major cause of senile dementia, mainly affecting elderly people over 65, but rarely early-onset Alzheimer's disease has also been reported. .

Patients suffering from Alzheimer's disease initially experience short-term memory loss, emotional ups and downs, loss of language skills, and long-term memory impairment, and eventually death. To date, no effective method for treating Alzheimer's disease is known, and the extent to which Alzheimer's disease can be prevented to some extent through exercise or balanced diet. In particular, in view of the domestic trend of rapid aging, there is a need to rapidly develop an agent that can effectively prevent and treat degenerative brain diseases including Alzheimer's disease, which is the main cause of senile dementia.

In relation to the cause of Alzheimer's disease, (i) hypothesis that it occurs when the synthesis of acetylcholine, a neurotransmitter, decreases (the acetylcholine hypothesis), and ii) the accumulation of amyloid peptides in the brain is the origin. (Amyloid hypothesis, iii) The hypothesis that Alzheimer's disease is initiated due to excessive phosphorylation of tau protein has been suggested, among which the amyloid hypothesis is widely supported.

 According to the amyloid hypothesis, as the most important cause of Alzheimer's disease, beta-amyloid (amyloid β peptide, Aβ), which consists of 40 to 42 amino acids, accumulates in brain cells, leading to oxidative damage of neurons that make up nerve cells. Finally, it is thought that it leads to dementia. Beta-amyloid is produced by proteolysis of amyloid precursor protein (APP). Three proteolytic enzymes, α-secretase, β-secretase and gamma-secretase (γ-SEC), are involved in the degradation of amyloid precursor proteins. Cretase) is involved.

More specifically, the amyloid precursor protein (APP) is a single trans-membrane protein, an extracellular N-terminal domain consisting of 590 to 680 amino acids and an intracellular signal. It consists of a cytoplasmic tail of about 55 amino acids, including an intracellular signal transduction sequence. The transcriptional RNA (mRNA) of the amyloid precursor protein gene forms eight isoforms through alternative splicing, and three of them are mainly present in the brain.

Amyloid precursor protein (APP) is largely subjected to proteolytic processing through two pathways. First, alpha-secretase cleaves the 17th amino acid of the beta-amyloid domain of the amyloid precursor protein to produce a relatively large water soluble N-terminal fragment (APPsα) and a non-amyloid C-terminal fragment (C83) of approximately 10 KDa. And these fragments are further degraded by gamma-secretase to produce non-amyloid peptides (P3). As another optional pathway, beta-secretase cleaves this precursor protein within the beta-amyloid domain of the amyloid precursor protein, resulting in approximately 100-KDa short aqueous soluble N-terminal fragments (APPsβ) By generating a C-terminal fragment (C99) of 12-KDa and further cleaving the C99 fragment by gamma-secretase, beta-amyloid is produced, depending on the site cleaved by gamma-secretase. Beta-amyloid (Aβ ₄₀ ) consisting of amino acids and beta-amyloid (Aβ ₄₂ ) consisting of 42 amino acids are produced.

Therefore, in order to prevent the accumulation of beta-amyloid (Aβ) in brain cells known to cause Alzheimer's disease, a representative degenerative brain disease, the amyloid precursor protein (APP) may be cut into amyloid-producing C-fragment (C99). There is a need to inhibit the activity of inducing beta-secretase.

Beta-secretase is also called BACE1 (β-site APP-cleaving enzyme 1) or memapsin 2. According to some studies to date, beta-secretase activity is increased by over-expression of transmembrane aspartic acid protease BACE1, and APP is cleaved by beta-secretase by over-expression of BACE1. It is precisely cleaved at the site, the activity of beta-secretase is inhibited by the BACE antisense oligonucleotide, and the purified BACE accurately cleaves APP and shows the properties of beta-secretase (Robert Vassar et al., β-Secretase Cleavage of Alzheimer's Amyloid Precursor Protein by the Transmembrane Aspartic Protease BACE, Science , 286, 735-741 (1999)) and aspartic acid protease, a membrane-binding protein, also produce beta-amyloid by beta-secretase. (Riqiang Yan et al, Membrane-anchored aspartyl protease with Alzheimer's disease β-secretase activity, Nature , 402, 533-537 (19 99).

In particular, beta-secretase is an enzyme that initiates beta-amyloid production by inhibiting the activity of beta-secretase, thereby preventing beta-amyloid reduction as well as preventing all subsequent harmful steps. -There are many advantages as a target for the treatment of Alzheimer's disease because it is clinically safe since there is no serious problem even if the gene encoding the secretase is modified. Regarding inhibitors of beta-secretase, hydroxyethylene-based substances having peptide bonds or compounds in which N-terminus is substituted with phenylacetyl groups are known. However, enzyme inhibitors having therapeutic activity should have a size of less than 700 Da. Because of its stable effects on metabolic processes in the body, continuous research is needed. In addition, chitosan derivatives, catechins, plant-derived phenolic compounds, and the like have been reported as inhibitors of beta-secretase as biologically-derived substances, but the clinical potential has not been reached.

Therefore, the present inventors studied the physiologically active function of the peptide obtained by hydrolyzing a protein that occupies a substantial portion of the components of the fish shells, which are mostly discarded fish products. As described above, as the consumption of aquatic products increases, various types of fish products have been developed and used. Accordingly, there is a large amount of by-products processed in the manufacturing process of fishery products (dark, skin, internal organs, fish bones, etc.), which requires efficient treatment of by-products. Most fish processing by-products are used or disposed of as feed, which makes it difficult to use resources efficiently and cause environmental pollution. However, by-products contain a large amount of useful components such as protein and collagen, and are highly applicable as a physiological functional material. Therefore, in the present invention, in order to efficiently use protein and collagen contained in fish shells efficiently, protein hydrolyzate was prepared by enzymatic hydrolysis, and the amino acid sequence was identified by separating and purifying an anti-Alzheimer's activity peptide from the hydrolyzate. In addition, the degree of beta-secretase inhibition, which is an index of anti-Alzheimer's activity, was confirmed in the state in which these separated and purified peptides were synthesized with low molecular weight peptides such as di-peptide to hexa-peptide.

The present invention will be described in detail as follows. Fish shells used in accordance with the present invention is a representative marine resource of the east coast squid ( Todarodes pacificus ) Shells and salmon ( Oncorhynchus keta ) shells and skates ( Raja Kenojei ) shells.

First, each of the salmon shell, the squid shell and the skate shell was crushed, and the hydrolyzate in the peptide state was obtained by using various proteolytic enzymes to hydrolyze the protein contained in these shells. Generally, a protein precipitation method using ammonium sulfate is known as a method of separating only proteins from body components of living organisms, but in this case, an additional process for protein immersion reaction time and desalting may be inefficient. Accordingly, in the present invention, the enzymatic hydrolysis method was used as a method of extracting the active substance from the skate skin.

Proteolytic enzymes used in accordance with embodiments of the present invention include alkalase, chymotrypsin such as α-chymotrypsin, neutrase, papain, pepsin Although trypsin was used, other inexpensive proteolytic enzymes, such as Protomax, Flavozyme, and other inexpensive proteolytic enzymes can of course be used.

At this time, it is necessary to adjust the maximum active pH, the activation temperature and the like of each enzyme for the activity of the proteolysis used, the same conditions as the optimal reaction conditions in the body of each protease can be applied. Conditions such as the maximum active pH for such a hydrolase are well known, and thus a detailed description thereof will be omitted. In this case, the protein hydrolase used and the body components of the skate skin used as a substrate may be mixed in a ratio of about 1:50 ~ 1: 200. According to an embodiment of the present invention, the hydrolysis efficiency of trypsin for protein hydrolysis in salmon shell body components, neutrase for protein hydrolysis in squid shell body components, and alkalase for protein hydrolysis in skate body components (See Example 1).

The degree of inhibition of beta-secretase activity, which initiates the synthesis of beta-amyloid, a major cause of Alzheimer's disease, was then analyzed in salmon, squid and skate skins. As mentioned above, according to the most widely supported amyloid hypothesis regarding the cause of Alzheimer's disease, the amyloid precursor protein (APP) is cleaved at a specific position by beta-secretase and then gamma-seated. Alzheimer's disease is caused by beta-amyloid produced by cleavage cleaved at different positions by accumulation in brain cells or neurons. Thus, in the present invention, the hydrolysis products using the respective enzymes for the protein in the body composition of salmon shells, squid shells and skate skins are hydrolyzed by the respective proteolytic enzymes in order to examine the effects of the treatment, prevention, etc. of Alzheimer's disease The extent to which the product inhibits the activity of beta-secretase was first analyzed. Interestingly, in relation to the inhibition of beta-secretase activity, among the protein hydrolysates derived from salmon shells, the hydrolysates by papain (see FIG. 1), and by pepsin from the protein hydrolysates derived from squid shells Among the hydrolyzate products (see FIG. 6), the hydrolyzate products by Neutrase (see FIG. 11) were good among the protein hydrolysis products derived from the skates.

Subsequently, the present inventors separated and purified an enzymatic hydrolysis product having good beta-secretase activity to obtain a peptide component exhibiting anti-Alzheimer's activity, thereby separating the peptide component having good beta-secretase activity and its amino acid sequence. Confirmed. Specifically, in the embodiment of the present invention, as a method for separating proteolytic products, which are crude extracts derived from fish shells, primary fractions are separated using column chromatography, wherein beta-secretase inhibitory activity is The second fraction components can be separated and purified using high performance liquid chromatography (HPLC) against the good primary fraction components. Finally, the amino acid sequence and molecular weight of the peptide having the inhibitory effect on beta-secretase activity were confirmed by MS / MS analysis. Of course, any other method that can separate and purify the crude extract in the form of peptides can be used.

According to an embodiment of the present invention, column chromatography is used to primaryly separate the protein hydrolysis products derived from salmon shells, squid shells and skate shells. In this case, as a support capable of fractionating peptides, which are protein hydrolysates derived from fish shells, by molecular weight, lipids, hormones, vitamins, and other small molecules in polar organic solvent or aqueous solvent mixtures may be used. Column chromatography using 'Sephadex G-25', an efficient column filler for the separation and extraction of compounds such as biomolecules, can be used.

According to an embodiment of the present invention, three primary fractions were obtained by column chromatography analysis of the protein hydrolyzate by papain derived from salmon shells, of which the first fraction was the most effective in inhibiting beta-secretase. (See Fig. 2), column chromatography analysis of the protein hydrolyzate by pepsin from squid shell yielded six primary fractions, the third of which had the highest beta-secretase inhibitory activity. (See FIG. 7), column chromatography analysis of the protein hydrolyzate by neutrase derived from skates yielded four primary fractions, of which the fourth fraction had the highest beta-secretase inhibitory activity. (See Figure 12).

On the other hand, the peptide component having an anti-Alzheimer's activity effect by analyzing the fraction component excellent in the beta-secretase inhibitory effect among the primary fraction components obtained through column chromatography analysis by high performance liquid chromatography (HPLC). (Secondary fraction component) can be separated and purified more specifically. In the case of salmon shells, HPLC analysis of the primary fractions with the best anti-Alzheimer's activity yielded eight secondary fractions, of which the third beta-secretase inhibitory activity was the best (FIG. In the case of squid skin, seven secondary fractions were obtained by HPLC analysis of the first fraction component having the best anti-Alzheimer's activity, and the beta-secretase inhibitory activity of the fifth fraction component was the highest. In the case of the skate, the primary fraction of the skates with the best anti-Alzheimer's activity was obtained by HPLC. Four secondary fractions were obtained, among which the beta-secretase of the second fraction was found. Inhibitory activity is best (see FIG. 13).

Subsequently, the fractions re-separated and purified through reverse-phase HPLC for the fraction component having the best beta-secretase inhibitory effect through HPLC analysis were compared to those of the second fraction component. The effect of inhibiting cretease activity is better (see FIG. 4 for salmon shell, see FIG. 9 for squid shell, and FIG. 14 for skate shell).

Then, the present inventors analyzed the amino acid sequence contained in the fraction which finally has anti-Alzheimer's activity using the MS / MS instrument. Peptides derived from skate skin are QGTRPLRGPEFL (Gln-Gly-Thr-Arg-Pro-Leu-Arg-Gly-Pro-Glu-Phe-Leu (SEQ ID NO: 1)), and peptides derived from salmon shells are ILFPL (Ile-Leu-Phe). Pro-Leu (SEQ ID NO: 12), squid shell-derived peptide (His-Val-Met-Leu, SEQ ID NO: 17), the lysine was commonly coupled to the C-terminus of the three purified peptides.

Then, the inventors of the present invention, the peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 17 described above only in order to prevent degradation by hydrolysis in vivo and improve the absorption rate and safety, only a part of the amino acid of the sequence A peptide consisting of was synthesized.

Specifically, with respect to the peptide derived from the skate shell (SEQ ID NO: 1), QGTR (Gln-Gly-Thr-Arg, SEQ ID NO: 2), QGT (Gln-Gly-Thr, SEQ ID NO: 3), QG (Gln-Gly, SEQ ID NO: 4), GTR (Gly-Thr-Arg, SEQ ID NO: 5), TR (Thr-Arg, SEQ ID NO: 6), PEFL (Pro-Glu-Phe-Leu, SEQ ID NO: 7), PEF (Pro-Glu- A peptide having the amino acid sequence of Phe, SEQ ID NO: 8), PE (Pro-Glu, SEQ ID NO: 9), EFL (Glu-Phe-Leu, SEQ ID NO: 10), FL (Phe-Leu, SEQ ID NO: 11) was synthesized . For peptides derived from salmon shells (SEQ ID NO: 12), ILF (Ile-Leu-Phe, SEQ ID NO: 13), IL (Ile-Leu, SEQ ID NO: 14), FPL (Phe-Pro-Leu, SEQ ID NO: 15), A peptide having the amino acid sequence of PL (Pro-Leu, SEQ ID NO: 16) was synthesized. For peptides derived from squid shells (SEQ ID NO: 17), HV (His-Val, SEQ ID NO: 18), ML (Met-Leu, SEQ ID NO: 19), HVM (His-Val-Met, SEQ ID NO: 20) and VML ( A peptide having the amino acid sequence of Val-Met-Leu, SEQ ID NO: 21) was synthesized.

As a result of measuring beta-secretase inhibitory activity using a peptide having an amino acid sequence from SEQ ID NO: 2 to SEQ ID NO: 21, in particular, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, The activity of the peptide consisting of the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21 was good. In particular, the anti-Alzheimer's activity of the peptide consisting of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21 was good, and among the peptides having leucine at the C-terminus, The activity of the peptide selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 21 was good.

In view of these results, the above-mentioned peptide derived from fish skin according to the present invention has anti-Alzheimer's activity, and thus can be used as an active ingredient in medicines or health foods.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of Alzheimer's disease containing the above-mentioned peptide as an active ingredient. Pharmaceutical dosage forms of the peptides according to the invention can also be used in the form of their pharmaceutically acceptable salts, and can also be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection. The composition for preventing and / or treating Alzheimer's disease related to the present invention may include the above-mentioned peptides derived from fish shells as an active ingredient alone, and according to other formulations, methods of use and purposes of use, that is, pharmaceuticals. It may further comprise an acceptable or nutritionally acceptable carrier, excipient, diluent or accessory.

Pharmaceutical compositions comprising peptides derived from fish skin according to the present invention, powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injections, respectively, according to a conventional method. It can be formulated and used in the form of a solution. For example, the peptide derived from the fish shell according to the present invention can be administered in various oral or parenteral formulations during clinical administration, and when formulated, it is commonly used fillers, extenders, binders, wetting agents, surfactants, anti- Diluents or excipients such as flocculants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included, and can be used orally or parenterally.

For example, carriers, excipients and diluents which may be included in pharmacological compositions comprising the peptides according to the invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, dextrins, calcium carbonate , Propylene glycol, liquid paraffin, may be one or more selected from the group consisting of physiological saline, but is not limited thereto, and any conventional carrier, excipient or diluent may be used. The active ingredient may be added to the peptide alone or in combination.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the peptides of the invention. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and glycerogelatin can be used. The pharmaceutical composition of the present invention may be via subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

In addition, the formulation of the composition for the prevention or treatment of Alzheimer's disease of the present invention may be in a preferred form depending on the method of use, and in particular in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Known methods may be employed to formulate. Exemplary formulations include, but are not limited to, excipients, granules, lotions, liniments, rimonadense, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, Suspensions, premixes, infusions, eye drops, tablets, suppositories, injections, main tablets, capsules, creams, pills, soft or hard gelatin capsules.

Furthermore, compositions for the prevention or treatment of Alzheimer's disease of the present invention may be prepared using methods known in the art or as disclosed in Remington's Pharmaceutical Science (), Mack Publishing Company, Easton PA. It may be preferably formulated.

In addition, the composition for the prevention or treatment of Alzheimer's disease according to the present invention, in addition to the above-mentioned active ingredient, nutrients, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective properties It may further contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.

The pharmacological composition for preventing or treating Alzheimer's disease may include a peptide derived from the fish shell according to the present invention with respect to the total weight of the composition at a concentration of 0.001 to 10.0 mg / ml. However, according to the method of use and purpose of using the pharmacological composition for the prevention or treatment of Alzheimer's disease of the present invention, the content of the active ingredient can be properly adjusted.

The composition for preventing or treating Alzheimer's disease and the antimicrobial composition of the present invention can be directly applied to animals including humans. The animal is a biological group corresponding to a plant, and mainly eats organic matter as nutrients, and means that digestion, excretion, and respiratory organs are differentiated, and preferably, vertebrates, more preferably mammals. Specifically, the mammal may be human, pig, cow or goat.

Fish-derived peptides according to the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Preferred dosages of the compositions for the prevention or treatment of Alzheimer's disease comprising the peptides of the present invention will vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and will be appropriately selected by those skilled in the art. Can be. However, for the desired effect, the peptide derived from the fish shell of the present invention may be used at a concentration of 0.001 to 10.0 mg / ml, preferably 0.005 to 5.0 mg / ml, more preferably 0.01 to 5.0 mg / ml. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, according to the present invention, a peptide derived from a fish shell that inhibits beta-secretase activity may be used in a food composition such as a dietary supplement for the prevention and improvement of Alzheimer's disease containing the peptide as an active ingredient. have. Examples of the food composition of the present invention include food, food additives, beverages or beverage additives. In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages, preferably gum or candy.

Therefore, the present invention relates to a dietary supplement for the prevention or improvement of Alzheimer's disease containing the aforementioned peptide as an active ingredient. Foods to which the peptide of the present invention may be added include, for example, various foods, beverages, gums, candy, tea, vitamin complexes, functional foods, and the like. Specifically, examples of foods that may be utilized in the peptide of the present invention include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, Drinks, teas, drinks, alcoholic beverages and vitamin complexes and the like, and includes all of the health food in the usual sense.

In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements. The food, beverage or food additive may be prepared by a conventional production method.

Functional food as used herein refers to the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food processed and designed to fully express the gymnastic function related to the living body, preferably the functional food of the present invention is capable of fully expressing the gymnastic function related to the prevention or improvement of oral disease to the living body Means food. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include functional beverages. The functional beverage according to the present invention can be used to control the bio-defense rhythm of the beverage group or beverage composition to which the added value is imparted so that the function of the beverage acts on the specific purpose by physical, biochemical, biotechnological, Means a beverage which has been designed so that the body control function related to restoration and the like is sufficiently expressed in the living body.

When the peptide derived from the fish shell according to the present invention is used as a food additive, the peptide may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The food composition comprising the peptide as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation thereof. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

It may also be added to foods or beverages for the purpose of preventing and ameliorating Alzheimer's disease. At this time, the amount of the peptide in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml. . However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like. The health functional beverage composition of the present invention is not particularly limited to other ingredients other than those containing the peptide as essential ingredients in the indicated proportions, and additional food supplement additive ingredients such as various flavoring agents or natural carbohydrates, such as ordinary beverages, may be used as additional ingredients. It may contain.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose and the like; Conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like, as well as sugar alcohols such as xylitol, sorbitol and erythritol. As natural or sweetening agents other than those mentioned above, natural flavoring / sweetening agents such as taumartin, stevia extract (eg Rebaudioside A, glycyrgin); Synthetic flavors / sweetening agents such as saccharin, aspartame and the like can be used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 10 g per 100 ml of the composition of the present invention. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition according to the invention.

In addition to the above, the peptides of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, carbonation agents used in alcoholic beverages, and the like. In addition, the food engineering composition according to the present invention may contain a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight, preferably 0.01 to 1.0 part by weight, per 100 parts by weight of the peptide of the present invention.

Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments, but the present invention is not limited by the following examples.

Example 1 Preparation of Enzymatic Protein Hydrolysates from Fish Shells

In the present embodiment, in order to use the protein and collagen of the fish skin was hydrolyzed using proteolytic enzymes, the degree of hydrolysis was measured. The raw materials of redfish ( Raja Kenojei ), salmon ( Oncorhynchus keta ) and squid ( Todarodes pacificus ) were used in Jeollanam-do and Jumunjin Port, Gangneung-si, respectively.They were stored in a freezer at 80 ° C. After fine grinding it was used for the experiment. Proteolytic enzymes include Alcalase (Novozyme, Denmark), Neutrase (Novozyme), α-chymotrypsin (Sigma-Aldrich, St. Louis, Mo. USA), papain (Sigma-Aldrich), trypsin (Sigma-Aldrich). Pepsin (Junsei, Japan) was used.

Put 1.0g of skate, salmon and squid shell into 100 mL of buffer, and react the pH and temperature according to the optimum condition of each enzyme for 1 hour. The reaction was time. After the reaction for 12 hours, the supernatant was collected by centrifugation, and the collected supernatant was lyophilized and used for measuring β-secretase activity. Table 1 shows the results of measuring the degree of hydrolysis of the hydrolase according to the present example. The degree of hydrolysis of trypsin hydrolyzate, neutrase hydrolyzate and alcalase hydrolyzate was high in salmon, squid and skate.

Hydrolysis Degree of Enzymatic Hydrolysates of Fish Shells
Degree of hydrolysis (%) Salmon shells Squid shell Skate Alcalase 51.03 ± 1.24 60.12 ± 3.01 79.61 ± 3.98 alpha-chymotrypsin 69.32 ± 1.64 75.34 ± 3.76 72.26 ± 3.61 Neutrase 52.81 ± 1.94 81.23 ± 4.06 79.23 ± 5.31 Papain 70.16 ± 1.54 62.78 ± 3.19 16.09 ± 1.28 Pepsin 74.63 ± 2.01 18.34 ± 0.91 67.54 ± 4.79 Trypsin 81.61 ± 2.12 63.17 ± 3.15 51.95 ± 2.60

Example  2: salmon shell Hydrolyzate  beta- Secretase  Inhibitory activity

In this embodiment, in order to confirm the prevention and treatment effect of Alzheimer's disease, which is a degenerative brain disease of the salmon skin-derived hydrolyzate obtained by treating the various proteolytic enzymes used in Example 1 above, these proteolytic products are Alzheimer's disease. The inhibitory activity of beta-secretase involved in the major mechanism of progression was analyzed by β-secretase inhibitory activity assay.

To this end, enzyme hydrolysates containing a large amount of peptides with high β-secretase inhibitory activity were selected from β-secretase inhibitory activity assays. Beta-secretase inhibitory activity was determined by modifying the method presented in the beta-secret ptase assay kit.

First, the enzyme hydrolysis products containing a large amount of peptides having high beta-secretase inhibitory activity among the proteolytic products were selected using this assay technique. Beta-secretase inhibitory activity was measured by modifying the method presented in the beta-secretase assay kit.

70 μl buffer (50 mM sodium acetate, pH 4.5), 10 μl β-secretase (1.0 Unit / mL), 10 μl substrate (10 mM MCA- [ASN670, LEU671] -AMYLOID BETA / A4 PRECURSOR PROTEIN 77, 50 mM sodium acetate buffer (pH 4.5) was mixed with 10 μl of sample solution and reacted at 25 ° C. for 20 minutes under dark conditions. The fluorescence reader (Bio-Rad) was measured at excitation 360 nm and emission 405 nm. The inhibition rate of beta-secretase used the following formula.

Inhibition (%) = [1-{(SS ₀ ) / (CC ₀ )}] × 100

C: Fluorescence value 20 minutes after the start of the reaction of the experimental group (Enzyme + buffer + substrate) without the addition of the sample solution

C ₀ : Fluorescence value of the zero time immediately before reaction of the experimental group (Enzyme + buffer + substrate) without the sample solution

S: Fluorescence value after 20 minutes of reaction of the experimental group (Enzyme + sample + substrate) to which the sample solution was added

S ₀ : Fluorescence value of the zero time immediately before the reaction of the sample group (Enzyme + sample + substrate)

Β-secretase inhibitory activity of each hydrolyzate derived from salmon skin was measured by IC ₅₀ value, and the results are shown in FIG. 1. Among hydrolyzate β-secretase inhibitory activity showed IC ₅₀ values of papain hydrolyzate as high as 0.83 ㎎ / ㎖ compared to other hydrolysates.

Example 3 Purification of Beta-Secretase Inhibiting Peptides from Salmon Shells

(1) column chromatography analysis

Beta-secretase inhibitory activity peptides were isolated and purified from papain hydrolyzate, which was found to have the best beta-secretase inhibitory activity among the protein hydrolysis products derived from salmon shells. The hydrolyzate derived from salmon shells digested with papain was dissolved in deionized water at a concentration of 100 mg / ml, and then injected into a column (2.5 × 75 cm) filled with Sephadex G-25 for gel filtration. The separation solvent was separated at a flow rate of 1.5 ml / min using deionized water, and the fraction was adjusted to 7.5 ml to measure the absorbance at 215 nm and separated into fractions. After lyophilization of each fraction, beta-secretase activity was measured. The measurement results are shown in FIG. As shown, three primary fractions (F1 to F3) were obtained from the papain hydrolyzate of the salmon shell, and the beta-secretase inhibitory activity of F1 was the best among the three fractions, as in Example 2 The IC ₅₀ value measured by the method was 0.75 mg / ml of F1.

(2) HPLC analysis

The fraction (F1) having the highest beta-secretase inhibitory activity among the primary fractions obtained above was subjected to high performance liquid chromatography using an ODS column (5 μm, 10 × 250 mm, YMC, Inc. Japan). chromatography, HPLC, Aglient 1100, Kyoto, Japan). Separation solvent was acetonitrile (0.1% trichloroacetic acid, JT Baker, USA) was separated at a flow rate of 2.0 ㎖ / min by a linear concentration gradient from 0% to 45% for 60 minutes. The separation results were measured at absorbance 215 nm and the measurement results are shown in FIG. 3. Eight secondary fractions (AH) were isolated as shown, and each fraction was lyophilized and beta-secretase inhibitory activity was measured as in Example 2. Beta-secretase inhibitory activity of each fraction was the best in fraction C with an IC ₅₀ value of 0.53 mg / ml.

(3) re-separation

As a result of HPLC analysis, the C fraction having the highest beta-secretase inhibitory activity among the second fraction components was re-separated and purified by reverse-phase HPLC (Agilent 1100) using an ODS column. For this purpose, an analytical column (particle size: 5 μm, 4.6 × 250 mm) was used to separate the conditions under a flow rate of 0.5 ml / min. The separation result was measured at absorbance of 215 nm, and the fraction was also subjected to beta-secretase inhibitory activity as in Example 2 after lyophilization. The measurement results are shown in FIG. 4, where the re-separation purification resulted in the separation of a single peptide of C-1 fraction, and the beta-secretase inhibitory activity of the final separation-purification fraction (C-1) may be expressed as an IC ₅₀ value. At that time, it was 0.07 mg / ml.

Example 4 Peptide Analysis from Salmon Shells

The amino acid sequence and the molecular weight of the active peptide of the isolated, purified fraction (C-1) determined to have the best effect were determined by HPLC and beta-secretase activity inhibition assay of Example 3. Amino acid sequencing was performed on nano-electron spectroscopy (ESI) quadrupole ion time-of-flight analyzer (TOF) mass spectrometers (Q-TOF Premier, sequentially connected to nano Ultra Performance Liquid Chromatography (nanoUPLC, Waters Corporation, Milford, Mass.). Waters Corporation, Manchester, UK). All assays were analyzed in cation mode. In this case, scans were performed at intervals of 0.6 seconds, and MS spectra was analyzed in the range of m / z 100 to m / z 1,600. At this time, among the ions having an ion detection intensity of 20 or more per second, three of the highest intensity were selected and received MS / MS spectra for 1.2 seconds in the range of m / z 50 to m / z 1,990 through an ion flight time difference analyzer. Data collection was analyzed by selecting ions with an ion detection intensity of 20 or more per second. The analysis results are shown in FIG. 5, and the finally analyzed peptide had five amino acid residues of ILFPL (Ile-Leu-Phe-Pro-Leu, SEQ ID NO: 12), and had a molecular weight of 609 Da.

Example 5 Beta-secretase Inhibitory Activity of Squid Shell Hydrolysates

The beta-secretase inhibitory activity was measured according to the same procedure as in Example 2 using hydrolysates derived from squid shells obtained by treating various proteolytic enzymes used in Example 1. The measurement results are shown in FIG. Among the hydrolyzates derived from squid shells, the beta-secretase inhibitory activity of pepsin hydrolyzate was the best and the IC ₅₀ value was 2.12 mg / ml.

Example 6: Purification of Beta-secretase Inhibiting Peptides from Squid Shells

(1) column chromatography

From the pepsin hydrolyzate of the squid skin with the best beta-secretase inhibitory activity according to Example 5, it was isolated by column chromatography using Sephadex G-25 column as in Example 3, and the beta-three for each fraction. Cretease inhibitory activity was measured. The measurement results are shown in FIG. 7, which were separated into six primary fractions (F1 to F6), of which the beta-secretase inhibitory activity of the F3 fraction was the best, with an IC ₅₀ value of 0.93 mg / ml. .

(2) HPLC analysis

F3 fraction, the first fraction component having the highest beta-secretase inhibitory activity, was analyzed by HPLC using the same column and HPLC equipment as in Example 3, and beta-secretase inhibitory activity was measured. Acetonitrile (0.1% trichloroacetic acid), which was a separation solvent, was separated at a flow rate of 2.0 ml / min by concentration gradient from 0% to 40% for 50 minutes, and the separation result was measured at absorbance of 215 nm. The measurement results are shown in FIG. 8, which were separated into seven secondary fractions (A to G), among which beta-secretase inhibitory activity of fraction E was the best, and the IC ₅₀ value of fraction E was 31.2 μg. / Ml.

(3) re-separation

For the second fraction E having the best beta-secretase inhibitory activity, beta-secretase inhibitory activity was measured after re-separation by reverse phase-HPLC as in Example 3 to obtain a fraction. Using an analytical column (particle size: 5 µm, 4.6 × 250 mm), acetonitrile (0.1% trichloroacetic acid), a separation solvent, was developed at a flow rate of 0.5 ml / min from 10% to 40% for 50 minutes. The separation result was measured at absorbance 215 nm. The measurement result is shown in FIG. 9, which was separated into a single peptide of the E fraction, and was 20.3 μg / ml when the beta-secretase inhibitory activity was expressed as IC ₅₀ .

Example 7 Peptide Analysis from Squid Shells

The amino acid sequence and molecular weight of the final fraction purified E in Example 6 were measured according to the same procedure and method as in Example 4. As shown in FIG. 10, the purified peptide had four amino acid residues of HVML (His-Val-Met-Leu, SEQ ID NO: 17), and had a molecular weight of 505 Da.

Example 8: Beta-secretase Inhibitory Activity of Skate Shell Hydrolysates

Beta-secretase inhibitory activity was measured according to the same procedure as in Example 2 using hydrolysates derived from skates obtained by treating various proteolytic enzymes used in Example 1. The measurement results are shown in FIG. Among the hydrolyzates derived from the skate skin, the beta-secretase inhibitory activity of the neutrase hydrolyzate was the best and the IC ₅₀ value was 0.56 mg / ml.

Example 9 Purification of Beta-Secretase Inhibiting Peptides from Skate Shells

(1) column chromatography

From the neutrase hydrolyzate of squid skin with the best beta-secretase inhibitory activity according to Example 8, it was isolated by column chromatography using Sephadex G-25 column as in Example 3, and beta- for each fraction. Secretase inhibitory activity was measured. Neutrase hydrolyzate diluted to 100 mg / ml in deionized water was injected into Sephadex G-25 column (2.5 × 75 cm), and deionized water, which was a separation solvent, was developed at a flow rate of 1.5 ml / min. The separated fractions were measured at absorbance 215 nm, and each fraction was lyophilized and a certain amount was taken to measure beta-secretase inhibitory activity. The measurement results are shown in FIG. 12, and were separated into four primary fractions (F1 to F4), among which the beta-secretase inhibitory activity of the F4 fraction was the best, with an IC ₅₀ value of 69.0 μg / ml. .

(2) HPLC analysis

F4 fraction, the first fraction component having the highest beta-secretase inhibitory activity, was analyzed by HPLC using the same column and HPLC equipment as in Example 3, and beta-secretase inhibitory activity was measured. F4 fraction with the highest β-secretase inhibitory activity was purified by HPLC (agilent 1100) using acetonitrile (0.1% trichloroacetic acid) as a separation solvent in an ODS column (5 μm, 10 × 250 mm, YMC, Japan) for 40 minutes. Concentration gradients from 0% to 50% were separated at a flow rate of 2.0 ml per minute, and the separation results were measured at absorbance 280 nm. The measurement results are shown in FIG. 13, which were separated into four secondary fractions (F1 to F4), among which beta-secretase inhibitory activity of fraction F2 was the best, and the IC ₅₀ value of fraction F2 was 51.3 µg. As / ml, the activity increased by 10.9 fold compared to the hydrolyzate before separation purification.

(3) re-separation

As for the second fraction F2 having the best beta-secretase inhibitory activity, beta-secretase inhibitory activity was measured after re-separation by reverse phase-HPLC as in Example 3 to obtain a fraction. Using an analytical column (particle size: 5 μm; 4.6 × 250 mm), acetonitrile (0.1% trichloroacetic acid) as a separation solvent was developed at a flow rate of 0.5 ml / min from 0% to 40% for 40 minutes. The separation result was measured at absorbance 280 nm. The measurement results are shown in FIG. 14, which were separated into a single peptide of F2-1 fraction, and the beta-secretase inhibitory activity of the F2-1 fraction was 32.5 μg / ml, represented by IC ₅₀ , and the hydrolyzate before repurification. 17.2 times increase.

Example 10 Peptide Analysis from Skate Shells

The amino acid sequence and the molecular weight of the fraction F2-1 finally separated and purified in Example 9 were measured according to the same procedure and method as in Example 4. The analysis results are shown in FIG. 15. The peptide finally purified according to this example had 12 amino acid residues of QGTRPLRGPEFL (Gln-Gly-Thr-Arg-Pro-Leu-Arg-Gly-Pro-Glu-Phe-Leu, SEQ ID NO: 1), The molecular weight was 1391 Da.

Example 11 Beta-Secretase Activity Assay of Final Purified Peptides

In this example, the peptide of SEQ ID NO: 12 derived from salmon shells analyzed in Example 4, the peptide of SEQ ID NO: 17 derived from squid shells analyzed in Example 7, and the sequence number 1 of skatefish shells analyzed in Example 10. In the peptide, the anti-Alzheimer's effect was examined by measuring the degree of inhibition of beta-secretase activity in the same manner as in Example 2. The measurement results are shown in Table 2 below.

Beta-secretase Inhibitory Activity of Purified Peptides Fish shells SEQ ID NO: Peptide amino acid sequence IC ₅₀ value (μM) Salmon shells 12 Ile-Leu-Phe-Pro-Leu 137.1 Squid shell 17 His-Val-Met-Leu 40.28 Skate One Gln-Gly-Thr-Arg-Pro-Leu-Arg-Gly-Pro-Glu-Phe-Leu 24.46

As can be seen from the measurement results in Table 2, peptides derived from salmon shells, squid shells and skate shells respectively increased the degree of inhibition of beta-secretase inhibitory activity compared to the hydrolyzate before purification. Accordingly, it was deduced from these purified peptides to look at the anti-Alzheimer's effect on peptides consisting of fewer amino acids than these peptides, for example those in the form of di-peptide to hexa-peptide.

Example 12 Amino Acid Synthesis and Activity of Anti-Alzheimer's Peptides

In Example 11 described above, a peptide composed of amino acids of the same or a part thereof was synthesized based on the amino acid sequence of three purified peptides having anti-Alzheimer's efficacy from fish shells. Anti-Alzheimer's activity was designed based on the amino acid sequence of the peptide to combine the N-terminal and C-terminal amino acid sequences to design new peptides with di-, tri- and tetra-peptides. Synthesis of three purified peptides and peptides having similar structures was synthesized using a solid phase peptide synthesis (SPPS) technique using a peptide autosynthesizer.

Peptide synthesis was synthesized in a peptide autosynthesizer (PeptrEx) using a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the Nα-amino group of amino acids. Specifically, the peptide having the carboxyl terminus of -NH ₂ form Rink Amide MBHA Resin as the initial material, and the peptide of the carboxyl terminus -OH form the Fmoc-amino acid-Wang Resin as the initial substance. Elongation of peptide chains by coupling of Fmoc-amino acids was performed by Nhydroxybenzo-triazole (HOBt) -dicyclo-hexylcar-bodiimide (DCC) method. After coupling the Fmoc-amino acid at the amino terminus of each peptide, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution and washed several times with NMP and dichloromethane (DCM), followed by nitrogen. Dry with gas. Trifluoroacetic acid (TFA) -phenolthioanisole-H ₂ O-triisop-ropylsilane (85: 5: 5: 2.5: 2.5, v / v) solution was added thereto and reacted for 3 hours to remove the protecting group and to separate the peptide from the resin. Next, the peptide was precipitated with diethyl ether. The precipitated peptide was purified using a C18 column (Delta Pak, C18 300 Å, 15 μm, 19.0 mm × 30 cm, Waters) in reverse phase-HPLC with acetonitrile concentration gradient containing 0.1% TFA.

The molecular weight was calculated based on the amino acid sequence of the synthesized peptide, and the exact molecular weight was determined using a matrix-assisted laser desorption ionization mass spectrometer. In addition, beta-secretase inhibitory activity was measured in the same manner as in Example 2 with respect to the synthesized peptide. Table 3 below shows the sequence of peptides derived from a skate and a portion of the peptide and the beta-secretase inhibitory activity of each peptide, and Table 3 shows the synthesis of amino acid sequences identical to those derived from skate. The amino acid sequence of the synthesized peptide consisting of the peptide and some amino acids and the result of measuring the beta-secretase inhibitory activity of each peptide are shown.

Beta-secretase Inhibitory Activity of Synthetic Peptides Based on Skate Shell-derived Peptides SEQ ID NO: Amino acid sequence of peptide IC ₅₀ value (μM) One QGTRPLRGPEFL 28.86 ± 1.44 2 QGTR 25.05 ± 1.25 3 QGT 23.17 ± 1.15 4 QG 26.21 ± 1.31 5 GTR 21.60 ± 1.08 6 TR 21.44 ± 1.07 7 PEFL 14.66 ± 1.75 8 PEF 111.72 ± 5.58 9 PE 23.03 ± 1.15 10 EFL 27.49 ± 1.37 11 FL 24.88 ± 1.24

On the other hand, Table 4 below shows the amino acid sequence of the synthesized peptide consisting of the same amino acid sequence as that derived from salmon shells and the peptide synthesized with some amino acids and beta-secretase inhibitory activity of each peptide.

Beta-secretase Inhibitory Activity of Synthetic Peptides Based on Peptides from Salmon Shells SEQ ID NO: Amino acid sequence of peptide IC ₅₀ value (μM) 12 ILFPL 126.77 ± 8.70 13 ILF 66.65 ± 0.52 14 IL 22.50 ± 4.88 15 FPL 11.71 ± 1.54 16 PL 18.68 ± 0.93

In addition, Table 5 below shows the amino acid sequence of the peptide composed of the same amino acid sequence as that derived from the squid shell and the peptide synthesized with some amino acids and the beta-secretase inhibitory activity of each peptide.

Beta-secretase Inhibitory Activity of Synthetic Peptides Based on Peptides from Squid Shells SEQ ID NO: Amino acid sequence of peptide IC ₅₀ value (μM) 17 HVML 44.52 ± 1.37 18 HV 34.37 ± 7.18 19 ML 5.62 ± 0.04 20 HVM 8.37 ± 1.55 21 VML 4.20 ± 0.21

In particular, it can be seen that the peptide consisting of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 21 has excellent beta-secretase inhibitory activity. In particular, among the peptides having leucine at the C-terminus, peptides of SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 are noted.

Attach an electronic file to a sequence list

Claims (6)

A pharmaceutical composition having a prophylactic or therapeutic effect of Alzheimer's disease, comprising as an active ingredient any of the peptides of SEQ ID NO: 21 in SEQ ID NO: 2.
The method of claim 1,
The peptides of SEQ ID NO: 12 to SEQ ID NO: 16 are hydrolysates or parts of the hydrolyzate treated with papain of the protein constituting the salmon shell, and the peptides of SEQ ID NO: 17 to SEQ ID NO: 21 constitute a squid shell A pharmaceutical composition having a prophylactic or therapeutic effect of Alzheimer's disease, characterized in that the hydrolyzate of pepsin-treated protein or part of the hydrolyzate.
3. The method according to claim 1 or 2,
The peptide is a pharmaceutical composition having a prophylactic or therapeutic effect of Alzheimer's disease having a beta-secretase (β-secretase) activity inhibitory effect.
3. The method according to claim 1 or 2,
The peptide is a pharmaceutical composition having a prophylactic or therapeutic effect of Alzheimer's disease is contained in the concentration of 0.001 ~ 10.0 mg / ㎖ in the pharmaceutical composition.
The method of claim 1,
A pharmaceutical composition for preventing or improving Alzheimer's disease, comprising a peptide having a leucine bound to the C-terminus as an active ingredient among the peptides.
A dietary supplement for the prevention or improvement of Alzheimer's disease, comprising as an active ingredient any one of the peptides of SEQ ID NO: 21 in SEQ ID NO: 2.

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