KR102382501B1 - Composition for preventing or treating alzheimer's disease of sea cucumber enzyme extract - Google Patents

Abstract

The present invention provides a use for the prevention, treatment or improvement of Alzheimer's dementia of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) enzyme extract. The enzyme extract according to the present invention exhibits the activity of beta-secretase (β-site Amyloid precursor protein Cleaving Enzyme, BACE), which is one of the enzymes involved in the production process of beta-amyloid peptide, which is considered to be the cause of Alzheimer's disease. By specifically inhibiting it, it can be usefully used as an active ingredient for preventing, treating or improving Alzheimer's disease.

Description

A composition for preventing and treating Alzheimer's dementia disease of sea cucumber enzyme extract {COMPOSITION FOR PREVENTING OR TREATING ALZHEIMER'S DISEASE OF SEA CUCUMBER ENZYME EXTRACT}

The present invention relates to the use of a composition comprising an enzyme extract of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) for preventing, improving or treating Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common neurological disorders and the most common age-related debilitating disease worldwide, leading to progressive amnesia, dementia and ultimately overall cognitive impairment and death. Currently, the only pharmacological therapies available are symptomatic drugs, such as cholinesterase inhibitors or other drugs used to control the secondary behavioral symptoms of AD. Research treatments targeting AD pathogenic process steps include therapies intended to inhibit the production, accumulation or toxic sequelae of beta-amyloid peptides (Non-Patent Document 1).

However, although synthetic drugs are useful for treating neurodegenerative diseases, researchers are paying attention to natural-source therapeutic agents due to related side effects (Non-Patent Document 2).

On the other hand, sea cucumbers are marine invertebrates belonging to the echinoderm family of sea cucumbers. They live mainly in coastal waters, and then migrate to deep open seas. The soft cylindrical sea cucumber is axially connected from the mouth to the anus. Sea cucumbers, which feed on decaying organic matter, play an important role in marine ecosystems. About 1,000 species are distributed worldwide, and about 40 of them are commercially available species and have medical significance (Non-Patent Documents 3 and 4).

In addition, various biologically active ingredients of sea cucumber have been widely used in Chinese folk medicine for centuries in China. Previous studies have investigated pharmacological and functional food aspects such as antioxidant activity (Non-Patent Document 5), antimicrobial activity, anticancer activity, anti-inflammatory activity (Non-Patent Document 6), and anticoagulant activity (Non-Patent Document 7) of sea cucumber has been proven.

Therefore, in the present invention, using the human neuroblastoma SH-SY5Y cell line, beta-site Amyloid precursor protein, one of the enzymes involved in the production of beta-amyloid peptide of sea cucumber in vitro Cleaving Enzyme, BACE) inhibitory activity was investigated.

V. P. Kramp and P. Herrling, Neurodegener. Dis., 2011 Cooper, E.L., Ma, M.J., 2017. Alzheimer Disease: Clues from traditional and complementary medicine. J. Tradit. Complement. Med. Xu, C., Zhang, R., Wen, Z., 2018. Bioactive compounds and biological functions of sea cucumbers as potential functional foods. J. Funct. Foods. Esmat, A.Y., Said, M.M., Soliman, A.A., El-Masry, K.S.H., Badiea, E.A., 2013. Bioactive compounds, antioxidant potential, and hepatoprotective activity of sea cucumber (Holothuria atra) against thioacetamide intoxication in rats. Nutrition. Esmat, A.Y., Said, M.M., Soliman, A.A., El-Masry, K.S.H., Badiea, E.A., 2013. Bioactive compounds, antioxidant potential, and hepatoprotective activity of sea cucumber (Holothuria atra) against thioacetamide intoxication in rats. Nutrition. Song, J., Li, T., Cheng, X., Ji, X., Gao, D., Du, M., Jiang, N., Liu, X., Mao, X., 2016. Sea cucumber peptides exert anti-inflammatory activity through suppressing NF-κand MAPK and inducing HO-1 in RAW264.7 macrophages. Food Funct. Mou, J., Wang, C., Li, W., Yang, J., 2017. Purification, structural characterization and anticoagulant properties of fucosylated chondroitin sulfate isolated from Holothuria mexicana. Int. J. Biol. Macromol.

The present invention aims to provide a composition for preventing, improving or treating Alzheimer's dementia by developing a substance capable of inhibiting beta-secretase, that is, preventing or treating Alzheimer's dementia, and including the same.

In addition, in the present invention, the step of extracting the sea cucumber ( Holothuria spinifera ) or the protruding sea cucumber (Stichopus japonicus ) with trypsin; And sea cucumber ( Holothuria spinifera ) or protrusion sea cucumber (Stichopus japonicus ) comprising the step of fractionating the extract by solid phase extraction (SPE) using a quaternary methylammonium (QMA) cartridge to obtain a fraction To provide a manufacturing method.

Accordingly, the present inventors have tried to find a natural material that can replace synthetic drugs as a substance having a preventive, ameliorating or therapeutic effect for Alzheimer's disease, sea cucumber ( Holothuria spinifera ) and protrusion sea cucumber (Stichopus japonicus ) by enzymes By extraction and fractionation, the present invention was completed.

In one embodiment, the enzyme extract of the sea cucumber ( Holothuria spinifera ) or the sea cucumber (Stichopus japonicus ) according to the present invention confirms the in vitro beta-secretase (β-site Amyloid precursor protein Cleaving Enzyme, BACE) inhibitory activity The IC ₅₀ value for the following was confirmed.

In addition, the sea cucumber ( Holothuria spinifera ) enzyme extract according to the present invention reduces the level of beta-secretase enzyme in the cell lysate of the SH-SY5Y neurocytoma cell line, thereby reducing the level of amyloid precursor protein (APP) confirmed to do.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising an enzyme extract of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) as an active ingredient.

The sea cucumber ( Holothuria spinifera ) or the protrusion sea cucumber (Stichopus japonicus ) of the present invention is not limited to its acquisition route and its site, but preferably the body wall of the sea cucumber may be used.

In one embodiment, sea cucumber ( Holothuria spinifera ) or protruding sea cucumber (Stichopus japonicus ) may be immersed in distilled water to remove sand and foreign substances, freeze-dried, and then pulverized to be hydrolyzed by an enzyme.

The enzyme may be at least one selected from the group consisting of alcalase, α-chymotrypsin, neutrase, papain and trypsin, preferably trypsin can be

In one embodiment, the enzyme extract of sea cucumber ( Holothuria spinifera ) or protrusion sea cucumber ( Stichopus japonicus ) is extracted by decomposing the sea cucumber with trypsin, and the extract is extracted using a quaternary methylammonium (QMA) cartridge. It may be a fraction obtained by fractionation by Solid Phase Extraction (SPE).

In the present invention, the sea cucumber ( Holothuria spinifera ) or the sea cucumber (Stichopus japonicus ) enzyme extract, hydrolyzate or fraction includes all extracts, fractions and purified products obtained in each step of fractionation or purification, dilutions, concentrates or dried products thereof. .

The food composition for preventing or improving Alzheimer's disease of the present invention, in particular, inhibits the activity of beta-secretase, and is used for the prevention or treatment of Alzheimer's dementia, a disease caused by the activity of beta-secretase can

The composition can be used together with the enzyme extract of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) according to the present invention, as well as the conventionally known Alzheimer's disease prevention or treatment agent.

The pharmaceutical composition comprising the enzyme extract of the sea cucumber ( Holothuria spinifera ) or the sea cucumber (Stichopus japonicus ) of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the extract, and the adjuvant includes Excipients, disintegrants, sweetening agents, binders, coating agents, expanding agents, lubricants, lubricants or flavoring agents and the like may be used.

The content of the enzyme extract of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) in the pharmaceutical composition can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, the administration method and the form of the preparation, etc., for example, the whole Based on 100 parts by weight of the composition, 0.0001 to 10 parts by weight or 0.001 to 1 parts by weight may be included, but is not limited thereto.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, extracts, injections, transdermal administrations, suppositories, and the like. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with a non-toxic, pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and color-developing agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pill, capsule, granule or tablet. Furthermore, by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the field, it can be preferably formulated according to each disease or component.

In addition, the present invention provides a food composition for preventing or improving Alzheimer's disease disease, comprising an enzyme extract of the sea cucumber ( Holothuria spinifera ) or protrusion sea cucumber (Stichopus japonicus ) as an active ingredient.

In the present invention, the term 'food composition' refers to an enzyme extract of the sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) added to food materials such as beverages, teas, spices, gum, confectionery, or encapsulated, powdered, or suspended. It is a food manufactured with food, etc., which means that it has a specific health effect when ingested.

In the present invention, the food composition for preventing or improving Alzheimer's dementia, like the pharmaceutical composition described above, may be used for preventing or improving Alzheimer's dementia caused by the activity of beta-secretase.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages , tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all foods in a conventional sense.

When using the enzyme extract of the sea cucumber ( Holothuria spinifera ) or the sea cucumber (Stichopus japonicus ) as a food additive, the sea cucumber ( Holothuria spinifera ) and the sea cucumber (Stichopus japonicus ) The enzyme extract is added as it is or together with other foods or other food ingredients. may be used, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be added within a range that does not impair the original taste of the food, and is usually 0.01 to 50 parts by weight, or 0.01 to 100 parts by weight, or 0.5 to 80 parts by weight, based on 100 parts by weight of the total composition for the target food. It can be added within the range. However, in the case of long-term intake for health and hygiene or health control, 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 above the above range. .

The food composition may further contain various flavoring agents or natural carbohydrates. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, or sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin or aspartame, and the like can be used. In addition to the above, the food composition according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the food composition of the present invention.

The present invention also provides a method for preventing or treating Alzheimer's dementia, comprising administering an enzyme extract of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) in a therapeutically effective amount to a mammal.

As used herein, the term 'mammal' refers to a mammal that is the subject of treatment, observation or experiment, and preferably refers to a human.

As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as conceived by a researcher, veterinarian, physician or other clinician, which an amount that induces amelioration of the symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other components contained in the composition, the type of formulation, and the age, weight, and general health of the patient It can be adjusted according to various factors including state, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. In the treatment method of the present invention, in the case of adults, the enzyme extract of the sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) of the present invention is administered once to several times a day, 1 mg/kg to 1000 mg/kg It is preferable to administer it in a dose.

In the treatment method of the present invention, the composition comprising the enzyme extract of the sea cucumber ( Holothuria spinifera ) or the sea cucumber ( Stichopus japonicus ) of the present invention as an active ingredient is oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal , transdermal, topical, intraocular or intradermal routes.

In one embodiment, the enzyme extract fraction of sea cucumber ( Holothuria spinifera ) or protrusion sea cucumber (Stichopus japonicus ) of the present invention is a sea cucumber (Holothuria spinifera ) or protrusion sea cucumber (Stichopus japonicus ) Extracting the extract with trypsin and quaternary of the extract It can be obtained by a manufacturing method through the step of obtaining a fraction by fractionation by solid phase extraction (SPE) using a methylammonium (QMA) cartridge.

In the present invention, the term 'enzyme extract of sea cucumber ( Holothuria spinifera ) or protrusion sea cucumber (Stichopus japonicus )' may be used as a term including the concept of fractions and hydrolysates.

The present invention will become apparent with reference to the embodiments which are described below in detail with their advantages and features, and methods of achieving them. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and is only defined by the scope of the claims of the present invention.

The enzyme extract of sea cucumber ( Holothuria spinifera ) or sea cucumber (Stichopus japonicus ) according to the present invention is effective for preventing, improving or treating Alzheimer's disease.

1 shows a process for separating and purifying beta-secretase inhibitory peptides from an enzyme extract of sea cucumber ( Holothuria spinifera ).
Figure 2 shows the yield of sea cucumber ( Holothuria spinifera ) enzyme extract. a indicates a significant difference within the column (p<0.05).
Figure 3 shows the IC ₅₀ value for the beta-secretase inhibitory activity of the sea cucumber ( Holothuria spinifera ) enzyme extract. a to b show significant differences within columns (p<0.05).
4 shows the IC ₅₀ values for the beta-secretase inhibitory activity fraction purified from ion exchange chromatography. a to c show significant differences within columns (p<0.05).
5 shows the HPLC chromatogram and reversed-phase HPLC of a potent beta-secretase inhibitory peptide purified from ion exchange chromatography. Fractionation was performed with a YMC Triart, C18 column with a linear gradient of 45% acetonitrile for 30 min at a flow rate of 1.0 ml/min. Fractionation was observed at (A) 215 nm and (B) 280 nm.
Figure 6 shows the morphological differences of the SH-SY5Y cell population. It can be seen that “S” type cells are epithelial without processes (arrows), whereas “N” type cells are more neuronal-like with a pyramidal body (arrowhead heads).
7 shows the effect of the hydrolyzate of sea cucumber ( Holothuria spinifera) on SH-SY5Y cell survival. Cell viability was compared with untreated control cells. a to b show significant differences within columns (p<0.05).
8 shows the effect of the F2 fraction purified from the trypsin hydrolyzate of sea cucumber ( Holothuria spinifera) on the viability of SH-SY5Y cells. Cell viability was compared with untreated control cells. a to b show significant differences within columns (p<0.05).
9 shows the effect of the F2 fraction purified from the hydrolyzate of sea cucumber ( Holothuria spinifera) on the BACE protein concentration in SH-SY5Y cells. a to c show significant differences within columns (p<0.05).
Figure 10 shows the effect of the F2 fraction purified from the hydrolyzate of sea cucumber ( Holothuria spinifera) on amyloid precursor protein levels in SH-SY5Y cells. a to c show significant differences within columns (p<0.05).
11 shows the effect of the F2 fraction purified from the trypsin hydrolyzate of sea cucumber ( Holothuria spinifera) on the sAPPβ protein level in SH-SY5Y cells. a to c show significant differences within columns (p<0.05).
12 shows the effect of the F2 fraction purified from the hydrolyzate of sea cucumber ( Holothuria spinifera) on the p-p38 protein level in SH-SY5Y cells. a to c show significant differences within columns (p<0.05).
Figure 13 shows the effect of the F2 fraction purified from the sea cucumber ( Holothuria spinifera) trypsin hydrolyzate on the p-JNK protein level in SH-SY5Y cells. a to c show significant differences within columns (p<0.05).

Hereinafter, the present invention will be described in detail through examples. The following examples only illustrate the present invention, but the scope of the present invention is not limited to the following examples.

[Example]

1. Materials and Methods

1-1. sample

Processed sea cucumber ( Holothuria spinifera) was purchased from a licensed exporter in Sri Lanka (Sugath International (Pvt) Ltd., Coly 15, Vystwyke Road, No. 65 / 352A), and Stichopus japonicus is located in Sokcho, East Coast. It was purchased from Baeknyeon Agricultural and Fisheries and used. The washed and weighed sea cucumbers were immersed in distilled water for 2 hours to remove salt and sand. The sea cucumber was then cut into small pieces and freeze-dried. The freeze-dried sea cucumber was ground into small particles and passed through a 600 μm aperture. Thereafter, the sea cucumber was stored at -80 ℃ until use.

1-2. Analysis of general components of dried sea cucumber

The general composition of the dried sea cucumber powder was determined according to the AOAC standard method (AOAC, 2005). The crude protein content of the ground sea cucumber dry powder was determined by an automated Kjeldahl system (Buchi, Flawil, Switzerland).

The percentage of Kjeldahl nitrogen was converted to protein content using a protein factor of 6.25. Ash content was measured by burning in a furnace (Thermolyne™) at 600° C. for 4 hours. Crude lipid content was measured using a Soxhlet extractor (VELP Scientifica, Milano, Italy). The moisture content of dried sea cucumber was measured by oven drying at 105° C. for 6 hours.

As a result, the general components of the dried sea cucumber ( Holothuria spinifera ) were found to be 79.77% crude protein, 14.80% carbohydrate, 2.96% ash, 1.96% lipid, and 0.78% moisture.

1-3. Amino Acid Analysis

To analyze the amino acid composition of sea cucumber ( Holothuria spinifera ), the sample was hydrolyzed with 6 N HCl at 110° C. for 24 hours. After hydrolysis, 5 mL of distilled water was added, and HCl was removed by evaporation to dryness at 45° C. under vacuum. After pre-derivatization with OPA and β-mercaptaethanol, centrifugation of the hydrolyzate using an Agilent 1100 HPLC system (Santa Clara, California, USA) and 20 μL of the filtered supernatant were used for amino acid analysis. . Amino acid separation was performed on a C18 column (5 μm, 4.6×250 mm, Waters, Massachusetts, USA). Amino acid quantitation was evaluated by comparison with retention times and peak areas of standard amino acids (Sigma Aldrich Co., St. Louis, USA).

As a result, the amino acid composition of the sea cucumber ( Holothuria spinifera ) dry powder showed the highest ratio of glutamic acid, alanine and aspartic acid at 178.89, 140.89, 94.07 and 93.14 g/kg, respectively, and the essential amino acid ratio was about 26.48%. .

1-4. Sea cucumber dry powder enzymatic hydrolysis

The enzymatic hydrolysis of sea cucumber dry powder contains alcalase, α-chymotrypsin (bovine pancreas, type II), neutrase, papain and trypsin (bovine Pancreas, type II) was performed under optimal temperature and pH conditions using five commercially available enzymes (Table 1). Hydrolysis was initiated at a 2% enzyme/substrate ratio and hydrolyzed at optimum temperature for 6 hours with shaking at 150 rpm. The hydrolysis reaction was terminated by inactivating the hydrolytic enzyme in the reaction solution by heating it at 95° C. for 10 minutes. Finally, the filtrate was lyophilized and stored at -80°C until use. The yield for each hydrolyzate after freeze-drying was calculated using Equation 1 below.

As a result, the yield was as high as 85.54% for α-chymotrypsin, 84.62% for trypsin, 74.50% for alcalase, 69.17% for papain, and 58.36% for neutrase. There was no significant difference between the yield obtained after hydrolysis and the yield of the five enzymes (FIG. 2).

[Formula 1]

Here, the dry weight W ₀ of the sample before hydrolysis is the dry weight of the sample after hydrolysis.

[Table 1] Optimal conditions for enzymatic hydrolysis of sea cucumber ( Holothuria spinifera ) or spiny sea cucumber ( Stichopus japonicus ) dry powder

1-5. Measurement of beta-secretase (β-site Amyloid precursor protein Cleaving Enzyme, BACE) inhibitory activity

Beta-secretase inhibitory activity was measured using a fluorescence resonance energy transfer (FRET) assay. To measure beta-secretase inhibitory activity, commercially available enzymes and peptide substrates were used. The peptide substrate contains a highly fluorescent 7-methoxycoumarin group that is efficiently quenched by resonance energy transfer to the 2,4-dinitrophenyl group. Thus, the substrate can be used to measure the activity of a peptidase capable of cleaving an amide bond between a fluorescent group and a quencher group that can cause an increase in fluorescence.

nnedorf, Switzerland)를 사용하여 96-well plate에서 수행되었다. The substrate contains a Swedish mutation of the amyloid precursor protein (APP) beta-secretase cleavage site. The beta-secretase inhibition assay was performed with an Infinite®200Pro Multimode Microplate Reader (Tecan, M) equipped with Tecan i-control software.

nnedorf, Switzerland) in 96-well plates.

First, 10 μL of beta-secretase (Sigma Chemical Co. (St. Louis, MO)) and 10 μL of a test sample were reacted at 70° C. in assay buffer (50 mM sodium acetate, pH 4.5) at 37° C. After 5 minutes, 10 μL substrate was added to 100 μL of the final volume, and fluorescence was measured at 340 nm and 430 nm for 2 hours at 5-minute intervals. Calculated.

[Formula 2]

where C ₀ is the initial fluorescence value (time = 0) of the control sample (enzyme, buffer, substrate), C is the fluorescence value of the control sample after 2 hours of incubation, S ₀ is the initial fluorescence value (time = 0) , S is the fluorescence value of the active sample after 2 hours of reaction.

As a result of measurement of beta-secretase inhibitory activity of the enzyme extract of sea cucumber ( Holothuria spinifera ), hydrolysates from α-chymotrypsin and trypsin had IC ₅₀ values of 66.80 μg/mL and 93.50 μg/mL, beta-secretase inhibition activity was quite high. Neutrase lysate and papain lysate exhibited beta-secretase inhibitory activity with IC ₅₀ values of 219.06 μg/mL and 202.70 μg/mL. Among the five hydrolysates, the Alcalase hydrolyzate having an IC ₅₀ value of 332.40 μg/mL significantly exhibited the lowest beta-secretase inhibitory activity ( FIG. 3 ).

The enzyme extract of Stichopus japonicus had the highest beta-secretase inhibitory activity with an IC ₅₀ value of 88.23 μg/mL of the trypsin hydrolyzate, and the IC ₅₀ values of alkalase, neutrase, and alpha chymotrypsin, respectively. Beta-secretase inhibitory activity was shown in the order of 93.86 μg/mL, 96.10 μg/mL, and 96.29 μg/mL (Table 2).

[Table 2] Beta-secretase inhibitory activity of enzyme extract of dried sea cucumber (Stichopus japonicus )

1-6. Purification on an ion exchange column

The trypsin hydrolyzate having the highest inhibitory activity against beta-secretase inhibition was separated with an ionic resin. Carboxymethyl (CM) and quaternary methyl ammonium (QMA) cartridges were used for separation.

The trypsin hydrolyzate was dissolved in distilled water to a concentration of 20 mg/mL and filtered using a 0.45 μm syringe filter. The CM cartridge was adjusted using 20 mM sodium phosphate buffer (pH 6.5) and equilibrated with distilled water.

The filtered sample was then loaded and the CM cartridge filtered fraction F1 was collected.

The QMA cartridge was stabilized using 20 mM Tris-HCL buffer (pH 8.0) and equilibrated with distilled water. The filtered sample was loaded into the cartridge and the filtrate F2 was collected. The F3 fraction was obtained through successive passes using CM and QMA cartridges. All three fractions were lyophilized and tested for beta-secretase inhibitory activity. The purification method is summarized in FIG. 1 .

As a result of purification, the main three fractions were obtained with yields of 65.02% for the F3 fraction, 77.58% for the F2 fraction, and 54.0% for the F3 fraction. The fraction F2 obtained after passing through the CM cartridge showed the highest beta-secretase inhibitory activity.

The IC ₅₀ value for the highest activity yield F2 was 75.50 μg/mL. The F1 fraction had the highest IC ₅₀ value of 1222.50 μg/mL, and the F3 fraction had an IC ₅₀ value of 1076.50 μg/mL with low activity compared to the hydrolyzate before purification ( FIG. 4 ).

1-7. Purification in Reverse Phase Chromatography

The ion exchange chromatography fraction with the highest beta-secretase inhibitory activity was analytical reversed-phase HPLC with a linear slope of acetonitrile (0-45% v/v) with 0.1% TFA in an HPLC system (Agilent Technologies, USA). A column (YMC-Triart C18, ø4.6×250 mm, 5 μm) was used for analysis. Elution was monitored at 215 nm and 280 nm on a diode array detector (DAD). The sample was divided into 4 fractions F2-F1, F2-F2, F2-F3, and F2-F4. Beta-secretase inhibitory activity was determined, and the fraction with the highest activity was purified by chromatography again on the same C18 column (FIG. 5).

As a result of purification, the F2-F4 fraction was fractionated in 100% acetonitrile, and the IC ₅₀ value was 78.97 μg/mL, showing the highest inhibitory activity. The F2-F3 fraction was fractionated with 33 to 45% acetonitrile, the IC ₅₀ value was 164.30 μg/mL, and the F2-F1 and F2-F2 fractions were fractionated from 15 to 24% to 25.5 to 31.5% acetonitrile. , showed lower inhibitory activity than 1200 μg/mL IC ₅₀ value.

1-8. Cell culture and differentiation

The human neuroblastoma SH-SY5Y cell line was purchased from the Korea Cell Line Bank (Seoul, Korea). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM): Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham (1:1) was mixed with heat inactivated fetal bovine serum (FBS) and 1% penicillin-streptate. Mycin was supplemented at 37° C., 5% CO ₂ .

Cell medium was changed every 2 days and subcultures of cells were performed at 90% confluence. All treatments were performed at ~80% confluence. For immunocytochemical MTT analysis, cells were aliquoted into 96 well plates at a density of 1 x 10 ⁴ per well. For Western blot analysis, cells were dispensed into a special well plate treated with tissue culture at a density of 5 x 10 ⁵ per well. Cells were differentiated by lowering FBS to 1% in DMEM/nutrient mixture F-12 Ham medium with 10 μM retinoic acid (RA). Morphological differences were observed between cell proliferation and differentiation under phase contrast light microscopy (Fig. 6).

1-9. MTT analysis

A modified method of the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay (Datki et al., 2003) was used. Cells were seeded in 96-well plates with Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham (1 : 1) supplemented with 1% penicillin-streptomycin at 37 °C, 5% CO ₂ for 24 h. . Cells cultured for 24 hours were washed twice with Dulbecco's phosphate buffered saline (DPBS). They were then treated with F1 fractions at concentrations of 50, 100, 200, 300 and 400 μg/mL. After 24 h of incubation, the cells were washed with DPBS and the medium was replaced with 100 μL MTT (5 mg/mL) solution. Again, the plate was reacted at 37 °C, 5% CO ₂ for 3 hours. The MTT solution was carefully removed and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formed formazan crystals. Color was measured at 540 nm with a Tecan F-200 Multi Microplate Reader (Tecan, Mannedorf, Zurich). Cell viability was calculated by the following formula.

As a result, concentrations of 50, 100, 200, 300 and 400 μg/mL had no cytotoxic effect on SH-SY5Y cells. More than 80% cell viability was observed at all five concentrations used for both hydrolyzate and F2 fractions (Figures 7 and 8). According to statistical analysis, only 400 μg/mL trypsin hydrolyzate showed a significantly lower cell viability compared to the control. However, the cell viability was higher than 80%. In the evaluation of the cytotoxicity of the F2 fraction, the concentration of 200 to 300 μg/mL significantly lowered the cell viability compared to the control sample. Since the cell viability was higher than 80% in both cases, five additional concentrations were used in the present invention.

[Formula 3]

In Equation 3, the abs _sample is the absorbance value of a well with treated cells, and the abs _blank is the absorbance of the well in MTT medium. Abs _control is the absorbance of wells that were not treated with cells.

1-10. Western blot analysis

For Western blot analysis cells in 6 well tissue culture plates were treated with F1 fractions at concentrations of 50, 100, 200, 300, 400 μg/mL for 24 h. At the end of 24 hours, the cells were washed with DPBS and incubated with trypsin EDTA at 37° C., 5% CO ₂ for 5 minutes. Suspended cells were collected, lysed with ice-cold lysis buffer (RIPA buffer and protease inhibitor cocktail) and homogenized for 1 min. Cells were centrifuged at 12,000 rpm for 10 min and the supernatant was collected. The protein content of the supernatant was determined using a BCA protein assay kit (Thermo Scientific, IL, USA). 15 μg of protein and 5x loading buffer were mixed in a microtube, heated at 95 °C for 5 min, and then loaded onto the gel. Depending on the molecular weight of the detection protein, the protein was separated on a 10% SDS polyacrylamide gel (a broad range of conserved protein markers ACCU-eco (Tech innovation, Kr). Proteins were separated by SDS-PAGE electrophoresis. Transfer to Ride (PVDF) membrane.The membrane is blocked with 5% nonfat skim milk in TBST (TBS containing 0.1% Tween-20) for 1 hour at room temperature.The membrane is incubated with primary antibody overnight at 4°C, Peroxidase secondary (HRP) antibody was incubated for 1-2 hours at room temperature.Finally, the protein band was detected with ECL detection reagent according to the manufacturer's instructions (Thermo Scientific, IL, USA).Blot was image lab Visualized in Chemidoc using the program (Bio-Rad Laboratories, CA, USA) Bands were normalized to beta-actin levels and band intensities were measured using Image Lab ^™ 6.0.1 analysis software.

Antibodies used were mouse-anti-amyloid beta (Aβ) (B-4): sc-28365, mouse anti-BACE (61-3E7): sc-33711 and m-IgGκ BP-HRP: sc-516102 (Santa Cruz, CA, USA.), rabbit anti-sAPPβ 813401 (biolegend, CA, USA).

- Statistical analysis

Data were analyzed in three independent experiments and results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was applied and comparisons between means were performed with Tukey's test and Duncan's comparison. Significant differences were measured at p < 0.05. Analysis was performed with SPSS 25.0 software (SPSS Inc., Chicago, IL, USA).

As a result of the analysis, the concentrations of 300 to 400 μg/mL treated with the fractions significantly reduced sAPPβ levels in SH-SY5Y cells.

In addition, treatment of sea cucumber ( Holothuria spinifera ) enzyme extract at a concentration of 50, 300 to 400 μg/mL significantly reduced p38 phosphorylation at the cellular level. In addition, treatment at concentrations of 50-400 μg/mL significantly reduced cell-level JNK phosphorylation by protecting neurons from apoptosis ( FIGS. 9-13 ).

Claims (11)

Contains an enzyme extract of sea cucumber ( Holothuria spinifera ) as an active ingredient,
The enzyme extract of the sea cucumber ( Holothuria spinifera ) is extracted by decomposing the sea cucumber with trypsin, and the fraction obtained by fractionating the extract by solid phase extraction (SPE) using a quaternary methylammonium (QMA) cartridge, Alzheimer's A pharmaceutical composition for preventing or treating sexually transmitted dementia. delete delete According to claim 1,
A pharmaceutical composition for preventing or treating Alzheimer's disease, wherein the enzyme extract of sea cucumber ( Holothuria spinifera ) is contained in an amount of 0.0001 to 10 parts by weight relative to 100 parts by weight of the pharmaceutical composition. According to claim 1,
A pharmaceutical composition for preventing or treating Alzheimer's disease, further comprising a pharmaceutically acceptable carrier. According to claim 1,
A pharmaceutical composition for preventing or treating Alzheimer's disease in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, extracts, injections, transdermal administrations or suppositories. Contains an enzyme extract of sea cucumber ( Holothuria spinifera ) as an active ingredient,
The enzyme extract of the sea cucumber ( Holothuria spinifera ) is extracted by decomposing the sea cucumber with trypsin, and the fraction obtained by fractionating the extract by solid phase extraction (SPE) using a quaternary methylammonium (QMA) cartridge, Alzheimer's Food composition for preventing or improving sexually transmitted dementia. 8. The method of claim 7,
The enzyme extract of sea cucumber ( Holothuria spinifera ) is a food composition for preventing or improving Alzheimer's disease disease, which is contained in an amount of 0.01 to 50 parts by weight relative to 100 parts by weight of the food composition. 8. The method of claim 7,
A food composition for preventing or improving Alzheimer's disease disease, further comprising a food-acceptable food supplement. delete delete

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