Hepatoprotective composition containing a mixture of enzymatic hydrolysate and acid hydrolysate of pig placenta

Abstract

The present invention relates to a composition for liver protection comprising a mixture of an enzyme hydrolysate and an acid hydrolysate of pig placenta, and a composition for preventing, improving or treating alcohol-induced liver damage, drug poisoning or hangover. The composition of the present invention has a significant effect on liver protection, particularly on preventing, improving or treating alcohol-induced liver damage, and can be usefully used in the fields of medicine and food.

Description

[Technical Field]

[0001] The present invention relates to a composition for protecting the liver, which comprises a mixture of an enzymatic hydrolysate and an acid hydrolysate of porcine placenta. [Background technology]

[0002] The liver is the organ in the human body where metabolism is most active, and various causes, such as excessive intake of fatty foods or alcohol, viral infection, harmful substances like various drugs, and nutritional deficiencies, can cause acute or chronic disorders, leading to fatty liver, hepatitis, jaundice, liver cirrhosis, and liver cancer. In particular, excessive intake of fat through food or excessive alcohol intake can lead to fatty liver, in which lipids accumulate in liver tissue, and this can cause increases in serum levels of AST (aspartate transaminase), ALT (alanine transaminase), and LDH (lactate dehydrogenase).

[0003] The placenta, composed of a blood chorion, maintains contact between the fetus and the mother's tissues, supplying the fetus with oxygen and nutrients. It also plays an important role in removing waste products produced by the fetus. Placenta contains various nutrients and hormones necessary for fetal growth. Pig placenta is widely used by adults, especially for the relief of menopausal symptoms and cosmetic purposes. Placenta contains essential amino acids, melatonin, nucleic acid components such as RNA and DNA, the antioxidant enzyme SOD (Super Oxide Dismutase), hyaluronic acid, antioxidants, cytokines, placenta peptides, insulin-like growth factors, epidermal growth factor (EGF), and senescent cell activating factor (SCAF), and other growth factors and cytokines, making it known to be useful for fatigue recovery and immune enhancement. Furthermore, among mammalian placentas, pig placenta has a high degree of homology with the protein structure of human placenta. It has been reported that pig placenta is a source of bio-active cytokines that control cell differentiation and fetal development, as it contains important components of proteins, various nutrients, DNA, and RNA. Due to these characteristics, pig placenta is used in food and pharmaceuticals. However, there is still insufficient research into pig placenta compositions that can effectively prevent or alleviate alcohol-related liver damage, drug poisoning, and hangovers. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a functional health food composition for liver protection, which contains a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient.

[0005] Another object of the present invention is to provide a functional health food composition for preventing or ameliorating alcohol-induced liver damage, drug poisoning, or hangover, which contains a mixture of an enzyme hydrolyzate and an acid hydrolyzate of pig placenta as an active ingredient.

[0006] It is still another object of the present invention to provide a pharmaceutical composition for protecting the liver, which comprises a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient.

[0007] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating alcohol-induced liver damage, drug poisoning or hangover, which comprises a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient. [Means for solving the problem]

[0008] To achieve the above object, the present invention provides a functional health food composition for liver protection, which contains a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient.

[0009] The present invention also provides a functional health food composition for preventing or ameliorating alcohol-induced liver damage, drug poisoning, or hangover, which contains a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient.

[0010] In one embodiment of the present invention, the porcine placenta enzymatic hydrolysate may contain one or more peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

[0011] In one embodiment of the present invention, the enzymatic hydrolysate and acid hydrolysate of pig placenta may be mixed in a weight ratio of 1:0.1-10, preferably 1:0.5-5, 1:0.6-5, 1:0.7-5, 1:0.8-5, 1:0.9-5, 1:1-5, 1:1-4, 1:1-3, 1:1, 1:2, 1:3, 1:4 or 1:5, but the ratio is not limited thereto.

[0012] In one embodiment of the present invention, the porcine placenta enzymatic hydrolysate may be prepared by treating with a protease, and the protease may be selected from the group consisting of papain, pronase, bromelain, and alcalase, but is not limited thereto.

[0013] In one embodiment of the present invention, the acid hydrolyzed pig placenta may be prepared by treating it with an acid, and the acid may be, but is not limited to, hydrochloric acid, sulfuric acid, acetic acid, or citric acid.

[0014] In one embodiment of the present invention, the peptide may be contained in the porcine placenta enzymatic hydrolysate at a concentration of 0.1 ppm to 100 ppm, preferably 1 to 25 ppm.

[0015] In one embodiment of the present invention, the mixture of the enzymatic hydrolysate and acid hydrolysate of the pig placenta may be contained in an amount of 1% to 20% by weight based on the total weight of the functional health food composition, and the peptide may be contained in a concentration of 0.001 ppm to 20 ppm in the total composition.

[0016] In one embodiment of the present invention, the composition reduces serum levels of ALP (alkaline phosphatase), AST (aspartate transaminase) or ALT (alanine transaminase).

[0017] The present invention also provides a pharmaceutical composition for protecting the liver, which comprises a mixture of an enzymatic hydrolysate and an acid hydrolysate of porcine placenta as an active ingredient.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating alcohol-induced liver damage, drug poisoning, or hangover, which comprises a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient.

[0019] In one embodiment of the present invention, the mixture of the enzymatic hydrolysate and acid hydrolysate of porcine placenta may be contained in an amount of 5% to 30% by weight based on the total weight of the pharmaceutical composition, and the peptide may be contained in a concentration of 0.005 ppm to 30 ppm in the total composition. [Effects of the Invention]

[0020] The composition according to the present invention has a significant effect on liver protection, particularly on the prevention, improvement or treatment of liver damage caused by alcohol, and can be usefully used in the fields of medicine and food. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows the HPLC patterns of enzymatic hydrolysates and acid hydrolysates of pig placenta. [Figure 2] LC / MS chromatogram of enzymatic hydrolysate of pig placenta. [Figure 3] Chromatogram results of pig placenta enzymatic hydrolysate and peptide (VVVE). [Figure 4] 1 shows the MS / MS patterns of pig placenta enzymatic hydrolysate and peptides (VVVE). [Figure 5] Chromatogram results of pig placenta enzymatic hydrolysate and peptide (DGLHLR). [Figure 6] 1 shows the MS / MS patterns of the pig placenta enzymatic hydrolysate and peptide (DGLHLR). [Figure 7] Chromatogram results of pig placenta enzymatic hydrolysate and peptide (DDFNPSVH). [Figure 8] 1 shows the MS / MS patterns of pig placenta enzymatic hydrolysate and peptide (DDFNPSVH). [Figure 9]The results show that alcohol dehydrogenase (ADH) activity in liver tissue was measured after administration of pig placenta enzymatic hydrolysate, acid hydrolysate, or a mixture of pig placenta enzymatic hydrolysate and acid hydrolysate (Normal: normal control group; Alcohol: negative control group; Silymarin: positive control group; L: low-dose pig placenta mixture group; M: medium-dose pig placenta mixture group; H: high-dose pig placenta mixture group; E-form: pig placenta enzymatic hydrolysate group; and A-form: pig placenta acid hydrolysate group). [Figure 10] The results show that aldehyde dehydrogenase (ALDH) activity in liver tissue was measured after administration of pig placenta enzymatic hydrolysate, acid hydrolysate, or a mixture of pig placenta enzymatic hydrolysate and acid hydrolysate (Normal: normal control group; Alcohol: negative control group; Silymarin: positive control group; L: low-dose pig placenta mixture group; M: medium-dose pig placenta mixture group; H: high-dose pig placenta mixture group; E-form: pig placenta enzymatic hydrolysate group; and A-form: pig placenta acid hydrolysate group). BEST MODE FOR CARRYING OUT THE INVENTION

[0022] In its best mode, the present invention provides a functional health food composition for liver protection, which contains a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient.

[0023] In addition, in its best form, the present invention provides a functional health food composition for preventing or ameliorating alcohol-induced liver damage, drug poisoning, or hangovers, which contains a mixture of pig placenta enzymatic hydrolysate and acid hydrolysate as an active ingredient. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides a functional health food composition for liver protection containing a mixture of pig placenta enzyme hydrolysate and acid hydrolysate as an active ingredient; and a functional health food composition for preventing or ameliorating alcohol-induced liver damage, drug poisoning, or hangovers.

[0025] The porcine placenta enzymatic hydrolysate may contain one or more peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

[0026] The peptide may be contained in the porcine placenta enzymatic hydrolysate at a concentration of 0.1 ppm to 100 ppm, preferably 1 to 25 ppm.

[0027] The term "porcine placenta enzymatic hydrolysate" as used herein refers to a porcine placenta enzymatic hydrolysate. of Protein hydrolase in It means that it has been processed and manufactured.

[0028] The proteolytic enzyme may be selected from the group consisting of, but not limited to, papain, pronase, bromelain and alcalase.

[0029] The term "porcine placenta acid decomposition product" as used herein refers to a porcine placenta with acid It means that it has been processed and manufactured.

[0030] The acid may be, but is not limited to, hydrochloric acid, sulfuric acid, acetic acid, or citric acid.

[0031] The enzymatic hydrolysate and acid hydrolysate of the porcine placenta may be mixed in a weight ratio of 1:0.1-10, preferably 1:0.5-5, 1:0.6-5, 1:0.7-5, 1:0.8-5, 1:0.9-5, 1:1-5, 1:1-4, 1:1-3, 1:1, 1:2, 1:3, 1:4 or 1:5, but is not limited thereto.

[0032] The functional health food composition of the present invention may include all foods in the usual sense, and may be interchangeably referred to as functional food, functional health food, or other terms known in the art.

[0033] The term "health functional food" as used herein refers to a food product manufactured and processed into the form of tablets, capsules, powders, granules, liquids, pills, etc., using ingredients or components with beneficial functions for the human body. Here, "functional" refers to the ability to regulate nutrients for the structure and function of the human body or to achieve beneficial health effects, such as physiological effects. The health functional food product of the present invention can be manufactured using methods commonly used in the art, and may be manufactured using ingredients and components commonly used in the art. Furthermore, the dosage form of the health functional food product of the present invention is not limited as long as it is recognized as a health functional food product. Unlike conventional medicines, the health functional food composition of the present invention has the advantages of being made from food ingredients and free from side effects that may occur with long-term drug use. It is also highly portable and can be taken as an adjuvant to promote the prevention or amelioration of alcohol-related liver damage, drug poisoning, or hangovers.

[0034] In the composition of the functional health food according to the present invention, the active ingredient (a mixture of the enzymatic hydrolysate and acid hydrolysate of pig placenta) may be contained in an amount of 1% to 20% (wt%) based on the total weight of the composition, but is not necessarily limited thereto, and the amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use, such as prevention, health, or treatment.

[0035] The dosage form of the health functional food can be any of powder, granule, pill, tablet, capsule, as well as general food or drink form.

[0036] The type of food is not particularly limited, and examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes, and may include any food in the usual sense.

[0037] Generally, when producing a food or beverage, the active ingredient can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for health and hygiene purposes or for health regulation purposes, the amount may be less than the above range, and since the present invention uses fractions from natural products, there are no safety issues and the active ingredient can be used in an amount greater than the above range.

[0038] The functional food according to the present invention may contain various flavorings or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates may be 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. Examples of sweeteners include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0039] In addition, the functional health food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated beverages. The functional health food composition of the present invention may also contain fruit pulp for producing natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. The proportion of these additives is not limited, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the functional food composition of the present invention.

[0040] The present invention also provides a pharmaceutical composition for liver protection containing a mixture of an enzymatic hydrolysate and an acid hydrolysate of pig placenta as an active ingredient; and a pharmaceutical composition for preventing or ameliorating alcohol-induced liver damage, drug poisoning, or hangover.

[0041] The pharmaceutical composition according to the present invention is not particularly limited in its content as long as it contains the active ingredient, but preferably, the active ingredient may be contained in an amount of 5 to 30 wt% based on the total weight of the composition. However, this is not limiting. Furthermore, the peptide may be contained in a concentration of 0.005 ppm to 30 ppm in the total composition. If the peptide is present in a concentration below this range, it may be difficult to achieve the desired preventive or therapeutic effect, and if the concentration exceeds this range, the expected effect may be insignificant.

[0042] The pharmaceutical composition according to the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injection solutions by conventional methods, and may contain suitable carriers, excipients, or diluents that are commonly used in the manufacture of pharmaceutical compositions for formulation.

[0043] The carrier, excipient, or diluent may be a variety of compounds or mixtures thereof, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0044] When the formulation is made, it can be produced using commonly used diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, surfactants, etc.

[0045] A solid preparation for oral administration can be prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to the excipient, a lubricant such as magnesium stearate or talc can also be used.

[0046] Oral liquid preparations include suspensions, solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin.

[0047] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerol gelatin.

[0048] The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition, body weight, severity of the disease, drug form, administration route, and administration period, and can be appropriately selected by those skilled in the art. However, to achieve desirable effects, the dosage may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg, per day. The dosage may be administered once a day or in divided doses. However, the scope of the present invention is not limited to the above dosage.

[0049] The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes, including oral, rectal, intravenous, intramuscular, subcutaneous, intracervical, or intracerebroventricular injection.

[0050] The present invention will be described in more detail with reference to the following examples. These examples are intended to explain the present invention in more detail, but the scope of the present invention is not limited to these examples.

[0051] Example 1. Production of porcine placenta enzyme hydrolysate

[0052] After thawing the pig placenta in a defroster, the placenta was cleaned of foreign matter with tap water and then placed in a meat kneader to facilitate deblooding. The placenta was then washed several times with 0.9% NaCl to remove any remaining blood. It was then crushed in a blender to facilitate hydrolysis. The prepared pig placenta was then hydrolyzed for 20 hours with 3% protease (papain). After hydrolysis, the protease was inactivated by heating. The pig placenta hydrolyzate was then contacted with a filter aid, filtered, and purified by adsorption. The pig placenta hydrolyzate filtrate was then added to 1.2x (w / w) ethanol and left for 15-20 hours. Insufficiently hydrolyzed sugars, proteins, and impurities were removed using a filter. The filtrate was concentrated and purified by adsorption using 0.1-2% activated carbon. The used activated carbon was then removed by filtration using a filter. The purified porcine placenta enzymatic hydrolysate from which activated carbon had been removed was filtered through a 0.2 μm filter and sterilized. Finally, a highly purified placenta extract was obtained.

[0053] Example 2. Production of acid decomposition product of pig placenta

[0054] After thawing the pig placenta in a defroster, 100 kg of pig placenta was purified with tap water and then decomposed in 70 kg of 35% (v / w) hydrochloric acid at 110°C for 22 hours, after which it was filtered. The filtered hydrolyzate was then concentrated and neutralized to pH 6-7 with sodium hydroxide, followed by adsorption purification using activated carbon. The activated carbon was removed by filtration. The purified pig placenta acid decomposition product with the activated carbon removed was then sterilized by filtration using a 0.2 μm filter.

[0055] Example 3. Analysis of enzymatic hydrolysate and acid hydrolysate of pig placenta

[0056] In order to confirm the characteristics of the enzymatic hydrolysate and acid hydrolysate of pig placenta, the present inventors conducted experiments to analyze the nitrogen content, amino acid content and HPLC pattern of the enzymatic hydrolysate and acid hydrolysate of pig placenta prepared.

[0057] As a result, the amino acid content of the enzymatic hydrolysate was approximately 40%, while that of the acid hydrolysate was approximately 80% (Table 1). In other words, it was confirmed that the acid hydrolysate had a higher amino acid content than the enzymatic hydrolysate. In addition, as shown in Figure 1, the HPLC patterns of the enzymatic hydrolysate and acid hydrolysate of pig placenta were also different (Figure 1).

[0058] [Table 1]

[0059]

number

[0060] Example 4. Peptide identification and efficacy analysis of enzymatic hydrolyzed pig placenta

[0061] 4.1. Mass Chromatography of Enzymatic Hydrolysate of Pig Placenta

[0062] 500 μL of methanol (MeOH) was added to 100 μL of the porcine placenta enzymatic hydrolyzate sample obtained in Example 1, and the mixture was vortexed and centrifuged. 600 μL of the supernatant was transferred to a new test tube and vacuum dried. Water was then added to the dried sample to make a total volume of 100 μL, and the filtered solution was subjected to sequence analysis using a MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255748 Germany) system and ms / ms ionization analysis.

[0063] <Analysis conditions> -Mobile phase: A mobile phase: H2O / FA=100 / 0.2(v / v) B mobile phase: Acetonitrile / FA=100 / 0.2(v / v)

[0064] [Table 2]

[0065] As a result of the analysis, a total of 17 peptides were analyzed, and 5 peptides were identified as porcine-derived peptides in the uniprot database (Table 3).

[0066] [Table 3]

[0067] Thereafter, in the present invention, three porcine-derived peptides were selected as indicator substances by checking the peak size and reproducibility among the five identified porcine-derived peptides (FIG. 2 and Table 4).

[0068] [Table 4]

[0069] Peptides having the same mass and ms / ms ionization form as the peptides identified from the pig placenta enzymatic hydrolysate were synthesized from Anygen (www.anygen.com) and used for the following experiments.

[0070] It was confirmed that the contents of the PEP-1, PEP-2 and PEP-3 peptides in the hydrolysate were 1 to 25 ppm.

[0071] 4.2. Peptide validation

[0072] Through verification of the PEP-1, PEP-2, and PEP-3 peptides, a peptide verification test was conducted to confirm that the peptides synthesized by Enygen were identical to the peptides present in the enzymatic hydrolysate of pig placenta.

[0073] In the case of PEP-1, peptide verification was performed using two methods. First, chromatograms of the pig placenta enzymatic hydrolysate and peptide (VVVE) were analyzed. Chromatograms were obtained by spiking the pig placenta hydrolysate (A), peptide (VVVE) (B), and synthetic peptide onto the pig placenta hydrolysate (C). As a result, the pig placenta hydrolysate and synthetic peptide were identified as identical peaks. This confirmed that the peptides matched the components present in the pig placenta hydrolysate (Figure 3). Second, MS / MS patterns of the pig placenta enzymatic hydrolysate and peptide (VVVE) were analyzed. As a result, the MS / MS patterns of the pig placenta hydrolysate and peptide (VVVE) matched, confirming that the peptides matched the components present in the pig placenta hydrolysate (Figure 4).

[0074] For PEP-2, the first method was to analyze the chromatograms of the pig placenta enzymatic hydrolysate and peptide (DGLHLR). The chromatograms were then examined using pig placenta hydrolysate (A), peptide (DGLHLR) (B), and pig placenta hydrolysate spiked with a synthetic peptide (C). The chromatograms confirmed the same peaks for the pig placenta hydrolysate and the synthetic peptide. This confirmed that the peptides matched the components present in the pig placenta hydrolysate (Figure 5). The second method for peptide verification was to examine the MS / MS patterns of the pig placenta enzymatic hydrolysate and peptide (DGLHLR). The MS / MS patterns of the pig placenta hydrolysate and peptide (DGLHLR) matched, confirming that the peptides matched the components present in the pig placenta hydrolysate (Figure 6).

[0075] For PEP-3, the first method was to analyze the chromatograms of the pig placenta enzymatic hydrolysate and peptide (DDFNPSVH). The chromatograms were then examined using pig placenta hydrolysate (A), peptide (DDFNPSVH) (B), and pig placenta hydrolysate spiked with a synthetic peptide (C). The pig placenta hydrolysate and the synthetic peptide were identified as having identical peaks. This confirmed that the peptides matched the components present in the pig placenta enzymatic hydrolysate (Figure 7). The second method for peptide verification was to examine the MS / MS patterns of the pig placenta enzymatic hydrolysate and peptide (DDFNPSVH). The MS / MS patterns of the pig placenta enzymatic hydrolysate and peptide (DDFNPSVH) matched, confirming that the peptides matched the components present in the pig placenta hydrolysate (Figure 8).

[0076] 4.3. Evaluation of peptide toxicity in HepG2 cells

[0077] After culturing HepG2 liver cancer cell line, three synthetic peptides were added at different concentrations, and then MTT (methylthiazol tetrazolium bromide, Sigma-Aldrich) was performed to confirm cell viability. An appropriate amount of cells (1 × 10) was added to each well of a 24-well plate (BD, Falcon). 5 The cells were seeded (1 / well) and treated with the respective concentrations of the samples. After 24 hours of incubation in a 37°C incubator, cell viability was confirmed. After 4 hours of incubation with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 400 μL of DMSO was added to each well to dissolve the insoluble formazan crystals. The absorbance was measured at 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0078] Cytotoxicity of the three peptides was confirmed, and all of PEP-1, PEP-2, and PEP-3 were non-toxic up to 10 μg / ml (Table 5).

[0079] 4.4. Hepatocellular protective ability of peptides

[0080] HepG2 cells, a liver cancer cell line, were plated in a 24-well plate at 1 × 10 5 The cells were divided into 1 / well plates and cultured. To confirm the hepatocellular protective ability of the three synthetic peptides, the cells were treated with various concentrations of peptides and cultured for 23 hours. To assess hepatocellular damage, the cells were treated with 10 mM t-BHP and cultured for 90 minutes. After incubation with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) for 4 hours, 400 μL of DMSO was added to each well to dissolve the insoluble formazan crystals. The absorbance was measured at 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0081] As a result, PEP-2 and PEP-3 showed high hepatocyte protection capacity of 26% and 20%, respectively, at 10 μg / ml (Table 5).

[0082] 4.5. Measurement of AST, a peptide-based liver function index

[0083] To confirm AST, each well (1 × 10 5Three synthetic peptides were added to the wells (1 / well) at varying concentrations and incubated for 23 hours. Then, 20 mM t-BHP was added and incubated for 3 hours. The supernatant was then measured using an Aspartate Transaminase (AST or SGOT) Activity Colorimetric Assay Kit (BIOVISION; K753-100). For cell quantification, the supernatant was removed and incubated with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) for 4 hours. Then, 400 μL of DMSO was added to each well to dissolve the insoluble formazan crystals. The absorbance was measured at 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0084] As a result, PEP-2 and PEP-3 showed the most significant AST suppression at 10 μg / ml, with values ​​of 34% and 14%, respectively (Table 6). As with the results of hepatocyte protection, PEP-2 was confirmed to have the most excellent efficacy.

[0085] 4.6. ALT measurement as a peptide-based liver function index

[0086] To confirm ALT, add 1 x 10 5Three synthetic peptides were added to each well (1 / well) at different concentrations and incubated for 23 hours. Then, 20 mM t-BHP was added and incubated for 3 hours. The supernatant was then measured using the Alanine transaminase (ALT or SGPT) Activity Colorimetric / Fluorometric Assay Kit (BIOVISION; K752-100). For cell quantification, the supernatant was removed and incubated with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) for 4 hours. Then, 400 μL of DMSO was added to each well to dissolve the insoluble formazan crystals. The absorbance was measured at 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0087] The results confirmed that PEP-2 had excellent ALT inhibitory activity, with the maximum efficacy observed at a concentration of 1 μg / ml, representing a value of 34% (Table 5).

[0088] [Table 5]

[0089] Example 5. In-vitro evaluation of the liver-improving efficacy of enzymatic hydrolysate and acid hydrolysate of pig placenta

[0090] The inventors conducted a cytotoxicity experiment of the enzymatic hydrolysate and acid hydrolysate of pig placenta by treating a human liver cancer cell line with the enzymatic hydrolysate and acid hydrolysate of pig placenta. Briefly, HepG2 human liver cancer cell line was cultured in a cell culture flask until it reached 80% confluence. 1.5 × 10 cells were placed in a 24-well plate. 5The cells were divided into 1000 cells. After 48 hours of culture, each test substance was treated at different concentrations and cultured for an additional 24 hours. After 4 hours of incubation with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 400 μL of DMSO was added to each cell to dissolve the insoluble formazan crystals. The absorbance was measured at a wavelength of 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0091] As a result, it was confirmed that the enzymatic hydrolysate and acid hydrolysate of pig placenta were non-toxic up to a total nitrogen concentration of 0.5 mg / ml (Table 6).

[0092] In addition, the inventors conducted an experiment to confirm whether the enzymatic hydrolysate and acid hydrolysate of pig placenta have a hepatoprotective effect. Briefly, HepG2 human liver cancer cell line was cultured in a cell culture flask and, when it reached 80% confluence, 1.5 × 10 cells were placed in a 24-well plate. 5 The cells were divided into 100 cells. After 48 hours of culture, each test substance was treated at different concentrations and cultured for 23 hours. Then, t-BHP (tert-Butyl hydroperoxide, 10 mM) was treated with the cells simultaneously with the samples for 1 hour and 30 minutes. After 1 hour and 30 minutes of simultaneous treatment, MTT was used to confirm the protective ability of the hepatocytes compared to the t-BHP-induced liver injury treatment group.

[0093] The results showed that treatment of hepatocytes with t-BHP generated a large amount of ROS, damaging the hepatocytes. However, treatment with the enzyme hydrolysate of pig placenta protected hepatocytes by approximately 40% at a concentration of 0.05 mg / ml of total nitrogen, and treatment with the acid hydrolysate of pig placenta protected hepatocytes by approximately 21% at a concentration of 0.05 mg / ml of total nitrogen (Table 6).

[0094] The inventors also conducted an experiment to confirm the hepatotoxicity-suppressing effects of the enzymatic hydrolysate and acid hydrolysate of pig placenta. After culturing HepG2 human liver cancer cell line in a cell culture flask, when the cell line reached 80% confluence, 1.5 × 10 cells were placed in a 24-well plate. 5 The cells were divided into 1000 cells. After 48 hours of culture, each test substance was treated at different concentrations and cultured for 23 hours. After 23 hours, the cells were simultaneously treated with t-BHP (20 mM) for 3 hours. After 3 hours, the supernatant was collected and the levels of AST (aspartate transminase) and ALT (alanine transminase), indicators of liver damage, were measured using an AST and ALT activity kit. The cell number was also determined by treating with MTT solution and normalized.

[0095] As a result, it was confirmed that when treated with the enzymatic hydrolysate of porcine placenta at a concentration of 0.1 mg / ml of total nitrogen, AST was suppressed by 91%, and when treated with the acid hydrolysate of porcine placenta at a concentration of 0.1 mg / ml of total nitrogen, AST was suppressed by 93%. In addition, when treated with the enzymatic hydrolysate of porcine placenta at a concentration of 0.1 mg / ml of total nitrogen, ALT was suppressed by 23%, but when treated with the acid hydrolysate of porcine placenta, no change was observed (Table 6).

[0096] [Table 6]

[0097] Example 6. Characterization of a mixture of enzymatic hydrolysate and acid hydrolysate of porcine placenta

[0098] In order to confirm the characteristics of mixtures of porcine placenta enzymatic hydrolysate and acid hydrolysate at different ratios, the inventors conducted an experiment in which porcine placenta enzymatic hydrolysate and acid hydrolysate were mixed in weight ratios of 1:1, 1:2, 1:3, and 1:4, and then the nitrogen content and amino acid content were analyzed.

[0099] As a result, it was confirmed that the amino acid content increased as the ratio of the acid hydrolysate of pig placenta increased (Table 7). In subsequent experiments, a weight ratio of 1:3 was selected for the mixture of the enzyme hydrolysate and acid hydrolysate of pig placenta, and efficacy evaluation was carried out at various concentrations, low, medium, and high.

[0100] [Table 7]

[0101]

number

[0102] Example 7. In vivo efficacy evaluation of porcine placenta enzymatic hydrolyzate, acid hydrolyzate, and a mixture of porcine placenta enzymatic hydrolyzate and acid hydrolyzate on alcoholic liver injury

[0103] 7.1. Sample preparation

[0104] The enzymatic hydrolysate, acid hydrolysate, and mixture of the enzymatic hydrolysate and acid hydrolysate of pig placenta were spray-dried to prepare powder samples. The nitrogen and amino acid contents of the powder samples were analyzed to confirm their properties (Table 8).

[0105] [Table 8]

[0106]

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[0107] 7.2.Animal husbandry

[0108] In this study, 7-8 week-old SD albino mice (approximately 250 mg) were purchased and subjected to a one-week quarantine and acclimation period. Healthy mice were then used after monitoring for changes in body weight and general health. During the experiment, all groups except for Group 1 were fed a radiation-sterilized Lieber Decallie Liquid Ethanol Diet ad libitum for four weeks. In the fourth week, alcohol was administered orally twice a week (Tuesdays and Thursdays) without separate drinking water. The temperature and relative humidity in the animal room were maintained at 23±2°C and 40-60%, respectively, with ventilation rates of 10-12 times per hour. Light was also adjusted to maintain a 12-hour light-dark cycle.

[0109] 7.3. Preparation of the Lieber DeCalie Liquid Ethanol Diet

[0110] The alcoholic diet was prepared as follows: 1) the required weight of powdered feed (132.28 g) and 67 ml of alcohol were placed in a beaker containing 821 ml of water; 2) after adding enough water, the mixture was thoroughly stirred to eliminate lumps; 3) water was added up to the 1 L mark; 4) the feed was thoroughly stirred, and then the mixture was placed in a blender and blended for 30 seconds; and 5) a feeding tube (120 ml) was used.

[0111] If liquid food is fed in a standard feeding box, it may overflow or stick to the animal's body, resulting in a large amount of food being lost. Furthermore, the surface area exposed to air is large, which can cause the highly volatile alcohol to evaporate and the food to be easily oxidized, which can have a significant impact on the experiment. Therefore, a liquid food feeding box was used in this experiment.

[0112] 7.4. Test Group

[0113] The test groups were as follows: (1) Normal control group (2) Negative control group: Alcohol diet (fed ad libitum for 4 weeks) + 30% alcohol (1.4 g / kg, PO) administered twice in the fourth week (3) Positive control group: Silymarin (100 mg / kg / day, PO) (Yang et al., 2015) plus 30% alcohol (1.4 g / kg, PO) administered twice at week 4. (4) High-dose group of a mixture of porcine placenta enzyme hydrolysate and acid hydrolysate (1:3): 2952 mg / kg / day, PO + alcohol diet (fed ad libitum for 4 weeks) + 30% alcohol (1.4 g / kg, PO) twice on the 4th week (5) Medium-dose group of a mixture of porcine placenta enzyme hydrolysate and acid hydrolysate (1:3): 1771 mg / kg / day, PO + alcohol diet (fed ad libitum for 4 weeks) + 30% alcohol (1.4 g / kg, PO) twice in the 4th week (6) Low-dose group of a mixture of porcine placenta enzyme hydrolysate and acid hydrolysate (1:3): 590 mg / kg / day, PO + alcohol diet (fed ad libitum for 4 weeks) + 30% alcohol (1.4 g / kg, PO) twice in the 4th week (7) High-dose group of porcine placenta enzymatic hydrolysate: 2511 mg / kg / day, PO + alcohol diet (fed ad libitum for 4 weeks) + 30% alcohol (1.4 g / kg, PO) twice in the 4th week (8) High-dose group of acid hydrolyzed pig placenta: 3282 mg / kg / day, PO + alcohol diet (fed ad libitum for 4 weeks) + 30% alcohol (1.4 g / kg, PO) twice in the 4th week

[0114] 7.5. Improves liver function after alcoholic liver damage

[0115] After approximately one week of pre-feeding, the inventors performed the four-week experiment according to the protocol. The test sample was prepared at a fixed dose and administered orally once daily. Alcohol diet was prepared according to a fixed formulation method and provided daily ad libitum. In the fourth week, the animals were orally administered two more doses at the fixed dose. After four weeks of testing, after a 12-hour fast, the animals were euthanized by cardiac blood collection. Blood was centrifuged at 3,000 rpm and serum was analyzed using an alcohol kit and an acetaldehyde kit for comparative analysis. Blood samples were also collected via cardiac collection for hepatotoxicity assessment. The blood was centrifuged at 10,000 rpm for 10 minutes and liver enzyme levels were measured to assess hepatotoxicity. Liver tissue samples were then taken to assess liver histology and intrahepatic ADH and ALDH enzyme activity. All results were presented using mean and standard error, and significance between test groups was verified using Student's t-test and ANOVA test.

[0116] In this invention, serum indices such as ALP, ALT, and AST, which represent liver disease, as well as albumin and total protein, which represent liver synthetic capacity, were measured to verify the efficacy of pig placenta on not only liver inflammation but also the overall liver condition of liver synthetic capacity. The measured values ​​for each serum indices are shown in Table 9.

[0117] As a result, serum albumin, along with total protein, was used as an index of liver synthesis capacity, and no significant differences were observed between the groups.

[0118] ALP (alkaline phosphatase) is distributed mainly in the liver, bone tissue, intestinal tract, white blood cells, etc., and increases in values ​​are mainly due to the liver and bone tissue. In the present invention, when test substances (silymarin, pig placenta extract) were administered, ALP tended to decrease compared to the negative control group, and it was confirmed that ALP decreased significantly in the medium dose group of the mixture of pig placenta enzymatic hydrolysate and acid hydrolysate, and in the pig placenta enzymatic hydrolysate.

[0119] ALT and AST are representative aminotransferases that indicate liver function. In the negative control group administered alcohol, ALT levels increased by about 3.5 times compared to the normal control group, while administration of the test substances (silymarin, pig placenta extract) confirmed a decrease compared to the negative control group. In particular, administration of a mixture of pig placenta enzymatic hydrolysate and acid hydrolysate resulted in a dose-dependent decrease in ALT levels, with the highest dose of the mixture of pig placenta enzymatic hydrolysate and acid hydrolysate showing the most significant decrease in ALT levels.

[0120] In addition, in the negative control group administered alcohol, AST levels increased significantly due to liver toxicity compared to the normal control group, and AST levels decreased significantly in all test groups except for the high-dose enzymatic hydrolysate test substance.In particular, when a mixture of porcine placenta enzymatic hydrolysate and acid hydrolysate was administered, AST levels decreased in a dose-dependent manner, just like ALT.

[0121] [Table 9]

[0122] Additionally, when the activity of ADH, an alcohol-metabolizing enzyme in liver tissue, was measured, ADH activity increased in the negative control group (0.85) administered with alcohol compared to the normal control group (0.37) (Figure 9). Furthermore, a tendency for ADH activity to decrease was observed in the following order: silymarin > low dose pig placenta mixture > medium dose pig placenta mixture > high dose pig placenta mixture. Consequently, ADH activity decreased more in the pig placenta enzymatic hydrolysate and acid hydrolysate groups than in the acid hydrolysate groups alone. However, enzyme activity significantly increased in the acid hydrolysate group (Figure 9).

[0123] In addition, the activity of ALDH, an alcohol-metabolizing enzyme in liver tissue, was measured. ALDH activity was increased in the negative control group administered alcohol compared to the normal control group, while the increased ALDH activity due to enzyme induction was generally blunted in the groups administered silymarin or pig placenta extract (Figure 10). A mixture of pig placenta enzymatic hydrolysate and acid hydrolysate inhibited the enzyme induction effect of alcohol in a dose-dependent manner, with the highest dose of pig placenta mixture showing the most pronounced inhibition of ALDH activity (Figure 10). The effects of pig placenta enzymatic hydrolysate and acid hydrolysate were similar (Figure 10). This means that the mixture inhibited ALDH activity more than the groups treated with either compound alone.

[0124] Production Example 1: Production of functional health food composition

[0125] A mixture of pig placenta enzyme hydrolysate and acid hydrolysate (mixed at a weight ratio of 1:3) was dissolved in distilled water at a concentration of 100 mg / 100 mL (0.1 wt%) or 15,000 mg / 100 mL (15 wt%), respectively. To the solution, liquid fructose (0.5 wt%), oligosaccharides (2 wt%), sugar (2 wt%), and salt (0.5 wt%) were added and water was added to the remaining amount, and the mixture was blended to homogenize and flash sterilized to produce a health functional drink.

[0126] Preparation Example 2. Preparation of pharmaceutical composition

[0127] 1 mg of a mixture of porcine placenta enzyme hydrolysate and acid hydrolysate (mixed at a weight ratio of 1:3) was dissolved in 5 ml of distilled water or saline and sterilized to prepare an injection. Alternatively, it was freeze-dried into a vial and prepared into a powder formulation. Capsules were prepared by filling gelatin capsules with 100 mg of porcine placenta hydrolysate, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate.

Claims

1. The composition contains a mixture of an enzymatic hydrolysate of porcine placenta and an acid-decomposed product of porcine placenta as active ingredients, and the enzymatic hydrolysate of porcine placenta and the acid-decomposed product of porcine placenta are mixed in a weight ratio of 1:0.1 to 1:10, The porcine placenta enzymatic hydrolysate is produced by treating porcine placenta with a protease, The acid hydrolyzed pig placenta is prepared by treating pig placenta with acid, The pig placenta enzymatic hydrolysate is a) adding a protease to the blood-removed porcine placenta and hydrolyzing it for 20 hours to obtain a hydrolysate; b) heating the hydrolysate to inactivate the protease, and then filtering the hydrolysate to obtain a porcine placenta hydrolysate filtrate; c) adding ethanol to the porcine placenta hydrolysate filtrate to remove impurities, and then concentrating the porcine placenta hydrolysate filtrate to obtain a porcine placenta hydrolysate concentrate; d) the porcine placenta hydrolysate concentrate is filtered and then sterilized by a production method comprising the steps of: The porcine placenta enzymatic hydrolysate contains peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 3, The pig placenta acid decomposition product is a) adding acid to the blood-depleted pig placenta and decomposing it at 110°C for 22 hours to obtain a pig placenta decomposition product; b) concentrating the porcine placenta degradation product and then neutralizing it to obtain a neutralized porcine placenta degradation product solution; c) adding activated carbon to the neutralized porcine placenta decomposition product liquid to purify the liquid, thereby obtaining a purified porcine placenta decomposition product liquid; d) the purified solution is filtered and then sterilized; The acid hydrolyzate of pig placenta contains 80% or more amino acids, The acid is hydrochloric acid, sulfuric acid, acetic acid, or citric acid. Composition of a functional health food for liver protection.

2. The composition contains a mixture of an enzymatic hydrolysate of porcine placenta and an acid-decomposed product of porcine placenta as active ingredients, and the enzymatic hydrolysate of porcine placenta and the acid-decomposed product of porcine placenta are mixed in a weight ratio of 1:0.1 to 1:10, The pig placenta enzymatic hydrolysate is a) adding a protease to the blood-removed porcine placenta and hydrolyzing it for 20 hours to obtain a hydrolysate; b) heating the hydrolysate to inactivate the protease, and then filtering the hydrolysate to obtain a porcine placenta hydrolysate filtrate; c) adding ethanol to the porcine placenta hydrolysate filtrate to remove impurities, and then concentrating the porcine placenta hydrolysate filtrate to obtain a porcine placenta hydrolysate concentrate; d) the porcine placenta hydrolysate concentrate is filtered and then sterilized by a production method comprising the steps of: The porcine placenta enzymatic hydrolysate contains peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 3, The pig placenta acid decomposition product is a) adding acid to the blood-depleted pig placenta and decomposing it at 110°C for 22 hours to obtain a pig placenta decomposition product; b) concentrating the porcine placenta decomposition product and then neutralizing the concentrated product to obtain a neutralized porcine placenta decomposition product solution; c) adding activated carbon to the neutralized porcine placenta decomposition product liquid to purify the liquid, thereby obtaining a purified porcine placenta decomposition product liquid; d) the purified solution is filtered and then sterilized; The acid hydrolyzate of pig placenta contains 80% or more amino acids, The acid is hydrochloric acid, sulfuric acid, acetic acid, or citric acid. A functional health food composition for preventing or improving alcohol-induced liver damage, drug poisoning or hangovers.

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