KR102102295B1 - Composition comprising compound K and decursinol for extending life span and stimulating differentiation of cells - Google Patents

Abstract

The present invention relates to a composition for promoting life and differentiation of cells containing compound K and decursinol as an active ingredient, and of the red blood cells in the blood of the compound K and deckersinol mixture of the present invention To prevent the reduction of erythrocytes by promoting the prolongation of lifespan and differentiation of erythrocyte cells, and confirming that the long-term preservation of blood is possible, the compound K and deckersinol mixture of the present invention is a pharmaceutical composition for improving or preventing erythropoiesis It is expected to be used for the development of blood preservative compositions as well as health functional foods.

Description

Composition comprising compound K and decursinol for extending life span and stimulating differentiation of cells}

The present invention relates to a composition for promoting life and differentiation of cells comprising compound K and decursinol as active ingredients.

Blood circulates through the blood vessels, providing oxygen and nutrients, and transporting waste products to be excreted through the kidneys. In addition, it is responsible for transport of hormones secreted by the endocrine organs, defense against external pathogens, and body temperature regulation. The blood is composed of plasma as a liquid component and blood cells as a cell component, and the blood cells are divided into red blood cells (RBC, erythrocyte), white blood cells (WBC), and platelets. Red blood cells carry oxygen molecules through a protein called hemoglobin (Hb, hemoglobin), and white blood cells are divided into granulocytes, monocytes, and lymphocytes, and react against external invasion. Platelets, along with proteins in plasma, play an important role in blood clotting. These blood corpuscles are produced in the bone marrow through hematopoiesis.

Aging is a change that accumulates in cells, tissues, organ systems, and individuals over time, and bone marrow with hematopoietic function also undergoes changes as it ages. In erythrocytes, erythrocytes gradually increase in size and shorten lifespan due to aging, and white blood cells show little change in their number, but the bacterial killing ability of granulocytes and the ability to move to the inflammatory site decrease, and the number of T lymphocytes decreases slightly. On the other hand, the number of platelets does not change with aging (Shin, M.G., 2014; Jung, C.W., 2010). Aging of the hematopoietic system causes a decrease in the function of the adaptive immune system, an increase in the incidence of autoimmune diseases, an increase in blood tumors, a decrease in hematopoiesis, and an increase in aging-related anemia.

Red blood cells are responsible for supplying oxygen to every corner of the body, so it can be said to be an essential ingredient. However, if there is a problem with the amount of red blood cells, diseases such as erythropoiesis, erythropoiesis and erythropoiesis are easily developed. Erythrocytosis refers to a condition in which the number of red blood cells decreases due to a decrease in the production of red blood cells due to damage to the bone marrow or abnormal differentiation, and rapid removal of red blood cells from the blood (Lang, KS, et al., 2005). Production and development are inhibited, and dizziness appears. Anemia is one of the typical erythropoiesis, in addition to pancytopenia, fanconi's syndrome, granulocyteopenia, and myelodysplasia. The causes of erythrocyte reduction vary widely, for example, erythropoietin production decline due to renal failure, red blood cell production disorders in the bone marrow such as aplastic anemia, cancer in leukemia or bone marrow, bone marrow. Causes such as decreased activity of bone marrow stem cells due to aging, or decreased activity of bone marrow cells due to side effects of chemo or radiation chemotherapy.

Treatment methods for erythropoiesis may vary depending on the cause. The treatment of anemia through the supply of iron and the supply of nutrients is relatively easy to access through diet. However, when the proliferation and differentiation of hematopoietic stem cells is the cause, it has been attempted to treat with growth factors, cytokines, dexamethasone, androgens, and estrogens, but in other disease syndromes It turns out that there are visible side effects. On the other hand, treatment with hematopoietic stem cells was attempted, but this process requires a lot of cytokines, which is problematic in economic terms.

The main active ingredient of ginseng called pronoun of healthy food is saponin, and saponin contained only in ginseng is called ginsenoside. These ginsenosides are known to have little physiological activity because they are highly insoluble in water in an inactive state, but are poorly absorbed in the intestine. However, when sugar is separated from the inactive form of ginsenoside and converted into an active form, it does not dissolve well in water, but increases absorption in the human body and exerts a physiological effect.

Compound K (compound K) is a final metabolite and bioactive substance of ginsenosides, and is known to be difficult to manufacture in large quantities because of its active ginsenosides.

Decursinol (decursinol) is one of the physiologically active ingredients of Angelica is known to have an analgesic effect. In addition, it has been reported to be effective in protecting against neurotoxicity, preventing dementia, and sepsis. Deckersinol is present in a very small amount in the root of Angelica but can be obtained in large quantities by hydrolyzing decursin or decursinol angelate, the main components of Angelica Angelica.

Accordingly, the present inventors used compound K, one of the ginseng saponin components, and decursinol, an extract component of Angelica, to confirm the effect of prolonging cell life and promoting division, and the compound K and deckersinol mixtures reduced the life of red blood cells. The present invention could be completed by confirming that the cells were prolonged and promote cell division.

Korean Patent Publication No. 2016-0072802, which is a prior art, describes the effect of extending the lifespan of cells of ginseng saponins containing compound K, promoting cell differentiation, and increasing the number of red blood cells, but the compound K and deckersinol mixture of the present invention The effect by is not described or implied at all. In addition, Korean Patent Publication No. 2017-0085641 describes the anti-inflammatory effect of ginseng extract and the treatment of malignant anemia using the same, but the life extension and cell differentiation effect of red blood cells by the compound K and decursinol mixture of the present invention are not at all. Not stated and mentioned.

Korean Patent Publication No. 2016-0072802, a composition comprising ginseng saponins as an active ingredient, published on June 23, 2016. Korean Patent Publication No. 2017-0085641, Composition for the production of metal nanoparticles, including ginseng extract and its use, published on July 25, 2017.

Jung, C.W., Cytopenia in elderly patients, The Korean Journal of Medicine, 78 (2), 185-188, 2010. Lang, K.S., et al., Mechanism of suicidal erythrocyte death, Cell Physiol. Biochem., 15, 195-202, 2005. Shin, M.G., Aging and impired hematopoiesis, J. Korean Med. Assoc., 57 (4), 334-340, 2014.

An object of the present invention is to provide a composition for promoting life and differentiation of cells comprising compound K and decursinol as active ingredients.

In addition, an object of the present invention is to provide a composition for blood preservation comprising compound K and deckersinol as active ingredients.

The present invention relates to a composition for promoting life and differentiation of cells comprising compound K and decursinol as active ingredients.

The composition may include compound K, deckersinol, surfactant, heat-treated soluble chitosan and basic amino acids.

The composition may be a compound K and decicinol mixed in a weight ratio of 1 to 20: 1 to 20.

The cells may be red blood cells or stem cells.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating erythropoiesis, comprising the composition and a pharmaceutically acceptable excipient.

The erythropoietin may be panicopenia, Pancorti syndrome, granulocytopenia, myelodysplasia, aplastic anemia, hemolytic anemia or iron deficiency anemia.

The present invention also relates to a dietary supplement for improving red blood cells, comprising the composition and a food-acceptable food supplement additive.

Another aspect of the present invention provides a composition for preserving blood, which includes compound K and decursinol as active ingredients.

The blood preservation composition may include compound K, decursinol, a surfactant, heat-treated soluble chitosan, and basic amino acids.

The composition for preservation of blood may be made to contain 0.02-50 μM of compound K in blood and 10-50 μM of decursinol.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for prolonging life and promoting differentiation of cells comprising compound K and deckersinol as active ingredients.

The cells may be red blood cells or stem cells.

The stem cells are cells having the ability to differentiate into various types of body tissues, and can be differentiated into various tissue cells, and thus can be applied to treatments such as regenerating damaged tissues, and may be embryonic stem cells or adult stem cells. You can. Preferably, it is adult stem cells, and more preferably hematopoietic stem cells.

The erythroid cell refers to cells that are in the process of maturation from erythroid progenitor cells, and refers to all cells that are in the process of differentiation from erythrocyte progenitor cells to erythrocytes. Preferably, red blood cell progenitor cells or red blood cell, but are not limited thereto.

The red blood cell progenitor cells mean all cells included in the red blood cell formation process except for the red blood cells that have been matured. In the bone marrow, hematopoietic stem cells capable of differentiation into various hematopoietic cells exist, and a part of the hematopoietic stem cells are differentiated into red blood cell progenitor cells whose direction of differentiation into the red blood cell system is determined. The most primitive identified as erythrocyte progenitor cells are the cell forming cell-erythroid (BFU-E), and the colony forming unit-erythroid (CFU-E), which is slightly differentiated. . After CFU-E, it differentiates into proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, or orthochromatic erythroblast, and then polychromatic erythrocytes. erythocyte) matures into red blood cells by denuclearization.

The compound K is one of ginsenosides, a saponin component of ginseng, and can be separated from ginseng extract as a final metabolite or bioactive material of ginsenosides.

The ginseng may be at least one selected from the group consisting of roots of wild ginseng, wild ginseng, cultured roots, ginseng ginseng, hwagisam, jeonchissam and ginseng. However, it is not limited thereto.

The ginseng extract can be obtained by extracting ginseng with one or more solvents selected from the group consisting of water, lower alcohols of C1-4, acetone, hexane, dichloromethane and ethyl acetate, and as lower alcohols of C1-4 Methanol, ethanol, propanol, isopropanol, butanol, and the like. It is preferably ethanol.

In addition, the ginseng extract may be a ginseng concentrate having a compound ratio of 50% or more by treating a hydrolase to increase the content of compound K. The hydrolase treatment can be repeated one or more times, and as the number of hydrolase treatments increases, a ginseng concentrate having a high content ratio of compound K can be obtained.

The hydrolase is pectinase, β-glycosidase (β-glucosidase), α-glucosidase, cellulase, celluclast, viscozyme, and the like. It is pectinase. However, it is not limited thereto.

The compound K can be obtained by fractionating ginseng extract by chromatography, and the chromatography is silica gel column chromatography, silica gel vacuum liquid column chromatography, HP-20 column Chromatography (HP-20 column chromatography), RP-18 column chromatography, LH-20 column chromatography, preparation reverse phase-high performance liquid chromatography (preparative reversed- Phase high performance chromatography, medium pressure liquid chromatography, high-performance liquid chromatography, and the like can be used.

The decursinol is one of the physiologically active ingredients of Angelica, and can be separated from Angelica extract.

The Angelica extract can be obtained by extracting Angelica with one or more solvents selected from the group consisting of water, lower alcohols of C1-4, acetone, hexane, dichloromethane and ethyl acetate, and as lower alcohols of C1-4 Methanol, ethanol, propanol, isopropanol, butanol, and the like. It is preferably ethanol.

In addition, the Angelica extract may be a fraction re-fractionated with water, a lower alcohol of C1-4 or a mixture thereof, and a lower alcohol of C1-4 may be methanol, ethanol, propanol, isopropanol, butanol, and the like.

Deckersinol can be separated by thermal decomposition by adding a basic solution to Angelica keiskei koidz, and then neutralizing with an acidic solution.

The basic solution may be KOH, NaOH or a mixture thereof, preferably KOH. However, it is not limited thereto.

The acidic solution may be HCl, HNO ₃ , H ₂ SO ₄ or mixtures thereof, preferably HCl. However, it is not limited thereto.

Meanwhile, the compound K or deckersinol of the present invention may be prepared according to a conventional method in the art, or may be prepared as a pharmaceutically acceptable salt.

The composition may include compound K, deckersinol, surfactant, soluble chitosan and basic amino acids.

The surfactant, heat-treated soluble chitosan and basic amino acid may be solubilizing agents for solubilizing the compound K and decursinol.

The solubilization of Compound K and Deckersinol comprises: a first step of preparing a primary mixture by mixing a surfactant with Compound K and Deckersinol; After adding ethanol to the primary mixture, a second step of mixing the heat-soluble soluble chitosan to prepare a secondary mixture; A third step of preparing a tertiary mixture by dissolving it by adding basic amino acid and purified water to the secondary mixture and adjusting the pH to 8.6; And, the fourth step of sterilizing the tertiary mixture; It can be made available through

The composition may be compounded by solubilizing compound K or decursinol, respectively, through the solubilization method.

The solubilization (solubilization) refers to a phenomenon in which the solubility of a substance insoluble in water increases, and the solubilization method may be used to increase the solubility of the compound K of the present invention, which is a poorly soluble substance, and deckersinol.

The first stage of the surfactant (sodium dodecyl sulfate), polyoxyethylene sorbitan monooleate (Tween-80), polyoxyethylene sorbitan monostearate (polyoxythylene sorbitan) monostearate, Tween-60, polyoxyethylene sorbitan monopalmitate, Tween-40, polyoxyethylene sorbitan monolaurate, Tween-20, Triton X-100 -100), and nonyl phenoxypolyethoxylethanol (nonyl phenoxypolyethoxylethanol, NP-40).

The surfactant may be added in an amount of 0.01% by weight or more relative to a poorly soluble material.

The heat treatment soluble chitosan in the second step is to increase the solubility of the water-soluble chitosan and increase the uniformity, i) dissolving the water-soluble chitosan in a solution having a pH of 1 to autoclave; And, ii) neutralizing the solution of step i) by adding a basic solution; Can be prepared through.

The pH 1 solution may be acetic acid. It is preferably 0.1M acetic acid.

The heat-treated soluble chitosan may contain 0.1 to 10% [w / v] based on the composition. When the content of heat-treated soluble chitosan is less than 0.1%, it is difficult to impart a dispersing power and does not exhibit a practical effect. When it exceeds 10%, it becomes rather less uniform due to excessive viscosity, and when production capacity increases, it is difficult to mix and economically It is not desirable.

The basic amino acid in the third step is utilized as a dissolution aid, and may be arginine, lysine, histidine, or a mixture thereof, preferably arginine or lysine.

The basic amino acid may contain 0.1 to 4% [w / v] based on the composition. When the content of the basic amino acid is less than 0.1%, it is difficult to assist solubility and there is no practical effect, and when it exceeds 4%, gelation may occur due to excessive viscosity, which is not economically desirable.

The composition may be a compound K and decicinol mixed in a weight ratio of 1 to 20: 1 to 20. Preferably, compound K and deckercinol are mixed in a weight ratio of 1 to 10: 1 to 10. When the weight ratio of the compound K and decursinol is out of range, it is not preferable because the effect of extending the lifespan of cells and promoting differentiation is reduced.

The composition provides a pharmaceutical composition for the prevention or treatment of erythropoiesis, comprising the compound K and deckersinol mixture and a pharmaceutically acceptable excipient.

The compound K and decursinol mixture may be added in an amount of preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, and most preferably 0.001 to 30% by weight based on the total weight of the total pharmaceutical composition. .

The pharmaceutical composition is formulated in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, liquid, aerosol, external preparation, suppository, and sterile injectable solution, respectively, according to a conventional method. You can. Carriers, excipients and diluents that may be included in the pharmaceutical composition 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 oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, sweeteners, acidifying agents, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the compound K and decursinol mixture of the present invention, for example, starch, calcium carbonate It is prepared by mixing sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, acidifying agents, etc. Can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween-61, cacao butter, laurin butter, and glycerogelatin may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and generally, dosages range from 0.01 to 2000 mg / kg / day. A more preferable dosage is 1-500 mg / kg / day. The administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to various mammals, such as rats, livestock, and humans. Any mode of administration can be envisaged, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrathecal epidural or cerebrovascular injection and skin application. Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even for a long period of time for prophylactic purposes.

The erythropoietin refers to a disease in which the number of erythrocytes is less than or equal to the normal person, and may be erythropoiesis, Pancorti syndrome, granulocytopenia, myelodysplasia, aplastic anemia, hemolytic anemia or iron deficiency anemia, It is not limited to this.

The composition provides a dietary supplement for improving red blood cells, comprising the compound K and deckersinol mixture and a food-acceptable food supplement additive.

The health functional food is preferably compound 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 10% by weight, based on the total weight of the health functional food compound K and decicinol mixture Can be added.

The health functional food includes tablets, capsules, pills or liquids, and as foods to which the extract of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, health Functional foods, etc.

Another aspect of the present invention relates to a composition for preserving blood comprising compound K and decursinol as active ingredients.

The composition may include compound K, deckersinol, surfactant, soluble chitosan and basic amino acids.

The surfactant, heat-treated soluble chitosan and basic amino acid may be solubilizing agents for solubilizing the compound K and decursinol.

The solubilization of Compound K and Deckersinol comprises: a first step of preparing a primary mixture by mixing a surfactant with Compound K and Deckersinol; After adding ethanol to the primary mixture, a second step of mixing the heat-soluble soluble chitosan to prepare a secondary mixture; A third step of preparing a tertiary mixture by dissolving it by adding basic amino acid and purified water to the secondary mixture and adjusting the pH to 8.6; And, the fourth step of sterilizing the tertiary mixture; Can be done through

The blood preservation can control the reduction of the number of red blood cells and the reduction of the function of red blood cells by reducing the cell damage of blood cells, preferably red blood cells.

The blood preservation composition may be such that the final concentrations of Compound K and Deckersinol in the blood are 0.02-50 μM Compound K and 10-50 μM Deckersinol. Preferably, the compound K is 0.02 to 20 μM and decursinol is 10 to 20 μM, more preferably, the compound K is 0.02 to 1 μM and deckercinol is 10 to 15 μM, and most preferably, the compound K is 0.02 μM and decker. The glow should be 14.28 μM.

The present invention relates to a composition for promoting life and proliferation and differentiation of cells containing compound K and decursinol as an active ingredient, wherein the compound K and deckersinol mixture of the present invention are used for red blood cells in the blood. It was confirmed that the prolongation of lifespan and the differentiation of red blood cell cells prevented the reduction of red blood cells and long-term preservation of blood.

Through this, it is expected that the compound K and deckersinol mixture of the present invention can be used for the development of a composition for preventing or treating erythropoiesis, a pharmaceutical composition for improvement and improvement, and a composition for preserving blood.

1 shows a single peak of compound K, and it can be seen that compound K was separated from the ginseng extract.
2 shows the solubilized compound K (A), deckersinol (B) and compound K and deckersinol mixture (C).
Figure 3 shows the blood preservation effect by the compound K and deckersinol mixture of the present invention, (A) is blood to which physiological saline is added, and (B) is a compound K and deckersinol mixture of the present invention It is the blood added.
Figure 4 shows the effect of promoting extracorporeal erythrocyte survival by the compound K and decursinol mixture of the present invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the contents introduced here are thorough and complete, and are provided to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Preparation of compound K>

Example 1-1. Production of primary CKS

The ginseng used in the present invention is wild ginseng and was purchased and used from the Simmanisan Ginseng Farming Association located in Hamyang-gun.

Goat Ginseng is removed from the air and loaded on a porous shelf at 121 ° C for 10 minutes using an automatic thermo-humidity control device filled with steam to allow moisture to fall downward to remove excess moisture while exposing it to steam under a pressure of 1.1㎏ / ㎠. Heat treatment. The heated ginseng ginseng was cooled to 90 ° C. or less while opening a vent valve and injecting air, and the same operation was repeated twice more to prepare active ginseng.

After 60 g of the active ginseng prepared as above was finely crushed into particles of 1 mm or less and dried, 300 ml of 80% [v / v] ethanol (alcohol) was added and heat was added to obtain an extract. The extract was used to remove solids using 500 µm particle filter paper to secure active ginsenosides.

The active ginsenoside obtained in the above was concentrated with a rotary evaporator to obtain an active ginsenoside concentrate. After preparing the active ginsenoside suspension of 0.23% [w / v] using 95% [v / v] ethanol, the obtained active ginsenoside concentrate is prevented from causing harmful bacteria due to contamination. In order to maintain the above 50 ℃ was stored.

  An aqueous solution (enzyme solution) containing 2.4% pectinase (trade name Rapidase, DSM food, Netherlands) is maintained at 50 ° C using a temperature automatic device, and a tube peristaltic pump is used. The active ginsenoside suspension was continuously injected into the enzyme solution dropwise at a rate of 50 ml / min. After centrifuging the mixture of the enzyme solution and the active ginsenoside suspension, the precipitate was collected. The collected precipitate was suspended with 95% [v / v] ethanol, filtered using a filter paper to obtain a filtrate, and concentrated to concentrate. (Named as 1st CKS) 1 ~ 1.4g was secured.

Example 1-2. Manufacturing of secondary CKS

The primary CKS prepared in Example 1-1 was suspended with 95% [v / v] ethanol to prepare 1.0% [w / v] primary CKS suspension and maintained at 50 ° C or higher.

 Using the primary CKS suspension, the secondary CKS was prepared by performing the pectinase treatment procedure of Example 1-1 in the same manner. At this time, it was confirmed that the secondary CKS was recovered to about 90% of the weight of the primary CKS used.

Example 1-3. Manufacturing of tertiary CKS

The secondary CKS prepared in Example 1-2 was suspended with 95% [v / v] ethanol to prepare a 1.0% [w / v] secondary CKS suspension and maintained at 50 ° C or higher.

 Using the secondary CKS suspension, a third CKS was prepared by performing the pectinase treatment procedure of Example 1-1 in the same manner. At this time, it was confirmed that the third CKS was recovered to about 80% of the weight of the used second CKS.

Example 1-4. Manufacturing of 4th CKS

The tertiary CKS suspension prepared in Example 1-3 was suspended with 95% [v / v] ethanol to prepare 1.0% [w / v] tertiary CKS suspension and maintained at 50 ° C or higher.

 Using the tertiary CKS suspension, the quaternary CKS was prepared by performing the pectinase treatment procedure of Example 1-1 in the same manner. At this time, it was confirmed that the fourth CKS was recovered to about 80% of the weight of the used third CKS.

Example 1-5. Ginsenoside type and content analysis

Changes in ingredients and contents of ginsenosides according to the treatment steps of Examples 1-1 to 1-4 were confirmed.

The CKS prepared for each step prepared in Examples 1-1 to 1-4 was suspended with 95% [v / v] ethanol to prepare a suspension of 1% [w / v]. High-performance liquid chromatography (HPLC) according to concentration gradient elution conditions of acetonitrile: water (4: 1 → 1: 0 [v / v]) using each suspension [Agilent C ₁₈ column , 4.6 × 250 mm, 5 μm particle size, flow rate 1 ml / min, UV detection: 203 nm] to analyze the changes in the composition and content of ginsenosides, and the results are shown in Table 1.

CKS Ginsenoside content (%) Compound K F2 Rd Rg3 1st CKS 52 2.69 34.4 2 2nd CKS 75 2 14 1.8 3rd CKS 85 1.5 4 0.5 4th CKS 92 0 0.1 0.1

As shown in Table 1, as the number of pectinase treatments increases, the content of compound K increases and the content of other types of ginsenosides decreases, so that the content of compound K through the manufacturing process is 90%. The above method for producing ginseng concentrate could be established.

Example 1-6. Preparation of compound K

After suspending the quaternary CKS prepared in Example 1-4 with 95% [v / v] ethanol, according to the concentration gradient elution conditions of ethanol: water (0: 1 → 9.5: 0.5 [v: v]) HP-20 column chromatography was performed to obtain 20 fractions. Each fraction was analyzed for the components and contents of ginsenosides using the same method as in Example 1-5, and the results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that a single peak of compound K appeared in the 14th fraction, and through this, it was found that compound K of a single material was separated.

<Example 2. Preparation of decursinol>

Example 2-1. Preparation of Angelica Angelica Extract

The Angelica used in the present invention was made of Korean and North Korean true Angelica.

The Angelica Angelica was finely crushed to 40 mesh or less and dried to have a moisture content of 5% or less. To the crushed dried Angelica Angelica, 95% [v / v] ethanol was added to make it 2-4 times the weight of the Angelica Angelica and reacted for 12 hours to obtain an extract. After removing solids using filter paper, evaporated to dryness to concentrate the primary Angelica I got water. 1 L of ethanol per 1 kg of primary concentrate was suspended and left at -20 ° C for 10 hours, and the resulting precipitate was removed by centrifugation to secure the supernatant, and the obtained supernatant was evaporated to dryness to 2 I made a tea Angelica concentrate. After suspending 50 ℓ of 60% [v / v] ethanol in the secondary Angelica concentrate, centrifugation was performed to separate only the 60% [v / v] ethanol layer. The separated 60% [v / v] ethanol solution was evaporated to dryness, concentrated, and then dissolved in 90% ethanol to take a supernatant to obtain the final Angelica keiskei koidz extract.

Example 2-2. Deckersinol Preparation

100 g of Angelica keiskei koidz extract prepared in Example 2-1 was suspended with 95% [v / v] ethanol, treated with 500 ml of 0.1N KOH for thermal decomposition, and neutralized with 57.5 ml of 6N HCl. The KCl precipitate formed in the neutralization process was removed by centrifugation, and the supernatant was concentrated until a small amount of ethanol remained and left at 4 ° C or lower. When the decicinol having low solubility in ethanol precipitates during standing, only the precipitate is separated through filtration, and the separated precipitate is washed twice with cold distilled water at about 0 to 4 ° C to prepare deckersinol.

At this time, it was confirmed that the prepared deckersinol had a purity of 99.9%.

<Example 3. Preparation of compound K and decicinol solubilized mixture>

A mixture obtained by solubilizing compound K and decursinol, which are poorly soluble components of the present invention, was prepared. At this time, the method described in Korean Registered Patent No. 10-1717672 of the present inventor was referenced.

Chitosan was dissolved in a 0.1 M acetic acid solution having a pH of 1 to prepare a 5% [w / v] chitosan solution, followed by autoclaving. After that, neutralization was performed again using 6N NaOH so that the pH was 7.0 to prepare a heat-treated soluble chitosan solution.

Put 0.1 g of polyoxyethylene sorbitan monooleate (Tween-80) into the mixture of compound K prepared in Example 1-6 and decicinol prepared in Example 2-2 After mixing thoroughly, 1 ml of 95% [v / v] ethanol was added and mixed. The mixing ratio of the mixture was adjusted according to the treatment conditions in Table 2 below. Here, 5 g of the heat-treated soluble chitosan solution prepared above was added and mixed, and 1.5 g of basic amino acid lysine or arginine was added and mixed with about 90 ml of purified water, and then adjusted to a pH of 8.6 using 6M HCl or 6M NaOH. Then, the total volume was added to 100 ml with purified water. The solution was subjected to autoclaving to volatilize the ethanol component with sterile operation to prepare a compound K and decicinol solubilized mixture. In addition, a single substance solution of compound K or decursinol was also prepared in the same manner, and compound K (A), decursinol (B), compound K and decursinol mixture (C) solubilized in FIG. The composition is shown.

<Example 4. Confirmation of the effect of compound K and deckersinol mixture on blood cells>

In the case of red blood cells, as aging progresses, the life span becomes shorter and the number decreases. Thus, in order to confirm the effect of the compound K and the deckersinol mixture of the present invention on blood cells, the compound K of the present invention and the deckersinol mixture were administered to the aged mice, and then the blood was analyzed to determine the effect on the blood cells. Confirmed.

After purchasing 6-week-old male SD (Sprague Dawley) rats and growing them in an animal room for up to 15 months, they were randomly divided into experimental groups in Table 2 below. In the normal control group, mice grown up to 15 months were used, and in the negative control group, 1 ml of physiological saline was injected once a day into mice grown up to 15 months, and in the rest of the experimental group, mice were grown up to 15 months. The composition corresponding to Table 2 was treated for 30 days by oral administration once a day. On the 31st, euthanasia was performed and blood was collected for blood analysis. Blood analysis was performed by requesting a specialized blood analysis organization, Green Cross Lab Cell, and the results are shown in Table 3 below.

Experimental group Processing method Normal control - Eumseong Control Saline Experiment group 1 1st CKS 1.25mg / kg Experiment group 2 Solubilized Compound K 0.5mg / kg Experiment group 3 Solubilized decursinol 0.5mg / kg Experimental group 4 Solubilized Compound K and Decincinol mixture at a 1: 1 weight ratio of 0.5mg / kg Experiment group 5 Solubilized Compound K and Decincinol in a mixture ratio of 1: 4 by weight 0.5 mg / kg Experimental group 6 Solubilized Compound K and Decker's Nori 0.5mg / kg of a mixture mixed in a 1:10 weight ratio Experiment group 7 Solubilized Compound K and Decincinol in a mixture ratio of 4: 1 by weight 0.5mg / kg Experiment group 8 Solubilized Compound K and Decincinol mixed in a ratio of 10: 1 by weight 0.5mg / kg

Experimental group RBC
(× 10 ⁶ / μl) WBC
(× 10 ³ / μl) Platelet
(× 10 ³ / μl) Hb
(g / ㎗) Hct Triglyceride
(Mg / ㎗) Normal control 7.0 6.5 1,150 15 55 480 Eumseong Control 7.0 6.6 1,140 14 54 500 Experiment group 1 9.12 6.65 1,160 16.9 57.8 200 Experiment group 2 9.2 6.5 1,100 17 58 220 Experiment group 3 7.5 6.5 1,130 15 54 400 Experimental group 4 8.5 6.65 1,140 16.5 55 250 Experiment group 5 8.5 6.5 1,160 16.6 55 250 Experimental group 6 8.0 6.5 1,150 16 55 270 Experiment group 7 9.0 6.5 1,120 16.5 55 250 Experiment group 8 9.0 6.5 1,120 17 58 200

As shown in Table 3, it was confirmed that the number of red blood cells (RBCs) in Experimental Group 1 and Experimental Group 2 was similar. In the case of Experimental Group 1, Compound K contained 52%, and the primary CKS of 1.25 mg actually contained Compound K of 0.65 mg, indicating that the throughput of Compound K was higher than in Experimental Group 2. This shows that Compound K has an effect of increasing the number of red blood cells, and it can be sufficiently predicted that Compound K separated into a single substance is more effective in increasing the number of red blood cells.

In addition, it was confirmed that in the case of experimental group 3 treated with deckersinol alone, the number of red blood cells did not increase significantly compared to the experimental group 2 treated with compound K alone, which stabilizes red blood cells rather than the activity of increasing the number of red blood cells. It can be seen that it is related to.

In the case of Experimental Group 4 to Experimental Group 8 treated with the compound K and deckersinol mixture of the present invention, the number of red blood cells was higher than that of the normal control group, but less than that of the experimental group 2 treated with Compound K alone, based on this, the red blood cells It was confirmed that it does not cause excessive increase of. In addition, in the case of triglyceride in the blood, it was confirmed that the values in experimental groups 4 to 8 were lower than those in the normal control group. Excessive increase in the number of red blood cells interferes with the flow of blood and raises the viscosity of the blood, causing problems, and the compound K and deckersinol mixture of the present invention also helps maintain homeostasis in vivo through control of the number of red blood cells and reduction of triglycerides. It was found to be.

<Example 5. Confirmation of the effect of stem cells of the compound K and decursinol mixture>

Stem cells extracted from human wisdom teeth were used to confirm the effect of stem cells of the compound K of the present invention and decursinol mixture.

While culturing the stem cells extracted from the wisdom tooth, the number of stem cells was measured by treating the compound K and decursinol mixture of the present invention, and the differentiation of the stem cells was confirmed. When treated with sinol alone, the time taken to double the number of stem cells (generational time) was shortened, and when the compound K and deckercinol mixtures of the present invention were treated, each generation was compared to the case of treating alone. It was confirmed that the space was shortened more.

In addition, as a result of confirming the extent of cell life extension through the degree of inhibition of the expression of β-galactosidase in the stem cells, β-galacto in the case of treating the compound K and deckersinol mixture of the present invention The expression of sidase was reduced the most.

Through this, it was found that the composition comprising compound K and decursinol of the present invention is excellent in promoting the differentiation of stem cells and prolonging the life of the cells.

<Example 6. Confirmation of blood preservation effect of compound K and decursinol mixture>

In order to confirm the blood preservation effect of the compound K and decursinol mixture of the present invention, blood is collected, and compound K and decursinol of the present invention are 20 nM in blood and 14.28 uM dekersinol in the collected blood. The changes in blood were observed with the naked eye while the blood was stored by adding as much as possible, and the results are shown in FIG. 3.

As shown in FIG. 3, in the case of the control group (A), the red blood cells are aggregated and precipitated, thereby causing the color of the blood to be pale, whereas in the case of adding the compound K of the present invention and the decursinol mixture (B), the red blood cells No precipitation was observed, and it was confirmed that the initial state of blood collection was maintained.

Through this, it was found that when the compound K and deckersinol mixture of the present invention was added to the blood storage pack, the storage stability of the blood was increased and the storage stability could be maintained for a long time.

< Example  7. Compound  K and Deckersinol  Confirm the effect of mixture increase in vitro red blood cell survival>

In the case of blood, it should be refrigerated for blood transfusion for 35 days after collection, and disposed of after 35 days. Therefore, the value of prolonged storage of blood can reduce the waste of blood, and increasing or maintaining the number of red blood cells is an important factor for blood transfusion. Accordingly, it was confirmed the effect of increasing the survival rate of red blood cells of the compound K and decursinol mixture of the present invention.

After the experimental white rats were anesthetized with ether, blood was collected from a large vein using a syringe treated with a CPD (citrate-phosphate-dextrose), an anticoagulant, and divided into small portions. Each sample was divided into blood, treated to a concentration as shown in Table 4 below, sealed, and stored at 4 ° C for about 60 days. During the storage period, the number of red blood cells in the blood was measured using a SIEMENS CBC counter, and the results are shown in FIG. 4 and Table 4 below. At this time, the survival rate of red blood cells in Table 4 represents the ratio of red blood cells in each elapsed day with the number of red blood cells in the blood as 100% before each sample treatment.

Elapsed time
(Work) Red blood cell survival rate (%) Solubilized compound K 20 nM and
Deckersinol 14.28μM mixture Solubilized Compound K 20nM  Solubilized Deckersinol 14.28μM 0 100 100 100 3 100 100 102 7 102 102 104 18 102 101 79 31 100 98 50 39 92 76 33 45 83 61 30 53 56 44 22 60 40 19 20

As shown in Table 4, when looking at the survival rate of red blood cells in the blood on Day 60, when the solubilized compound K and decursinol mixture were treated, it was 40%, whereas when the solubilized compound K was treated alone, 19 %, And when treated with solubilized decursinol alone, about 20%, it was found that the compound K and decursinol mixture of the present invention has an excellent effect of enhancing the survival rate of red blood cells in vitro.

In addition, as shown in Figure 4, as a result of comparing the survival rate of in vitro red blood cells according to the processing time of each sample, when treated with solubilized decursinol, the level of the survival rate of red blood cells in the blood (no treatment) with no treatment was almost the same level. On the other hand, it showed that the survival rate of red blood cells was higher than that of solubilized decursinol or no treatment, especially when solubilized compound K alone or solubilized compound K and decursinol mixtures were treated. In the case of the solubilized compound K and deckersinol mixture, it was confirmed that the survival rate of red blood cells was the highest.

Although the specification of the present invention was not shown directly, in the case of the compound K and decursinol mixture, the survival rate of red blood cells in the blood is high when the compound K in the blood is included in the range of 0.02-50 μM and decursinol 10-50 μM. It was confirmed that it appeared.

Through this, it was found that when the compound K and deckersinol mixture of the present invention was added to the blood storage pack, the preservation of blood and the long-term preservation were possible while increasing the survival rate of red blood cells.

< Formulation example  1. Pharmaceutical preparation>

Formulation Example 1-1. Preparation of tablets

200 mg of the compound K and the decicinol solubilized mixture of the present invention were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid, respectively. After adding 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. The mixture obtained by drying it was added to 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate to form a tablet.

Formulation Example 1-2. Preparation of injection liquid

The compound K of the present invention and decicinol solubilized mixture 100mg, sodium chloride 0.6g and ascorbic acid 0.1g was dissolved in distilled water to make 100ml. The solution was put into a bottle and sterilized by heating at 20 ° C for 30 minutes.

<Formulation Example 2. Food manufacturing>

Formulation Example 2-1. Cooking seasoning

The compound K and decicinol solubilized mixture of the present invention were added to each cooking seasoning in 1% by weight to prepare a cooking seasoning for promoting health.

Formulation Example 2-2. Production of flour food

Compound K and deckerinolic solubilizing mixture of the present invention was added to flour at 0.1% by weight, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare health foods.

Formulation Example 2-3. Preparation of soup and gravy

The compound K of the present invention and the decicinol solubilized mixture were added to the gravy at 0.1% by weight to prepare health-enhancing soup and gravy.

Formulation Example 2-4. Manufacturing dairy products

Compound K of the present invention and decicinol solubilized mixture were added to milk at 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice production

Compound K and deckerinolic solubilized mixture of the present invention were added to tomato juice or carrot juice, respectively, to prepare a vegetable juice for health promotion.

Formulation example  2-6. Fruit juice manufacturing

0.1 g of each of the compound K and decicinol solubilized mixture of the present invention was added to 1,000 ml of apple juice or grape juice to prepare a fruit juice for health promotion.

Claims (10)

A composition for prolonging life of red blood cells comprising compound K and decursinol as active ingredients. In claim 1,
The composition is a composition for extending the life of red blood cells, characterized in that it comprises compound K, deckersinol, surfactant, heat-treated soluble chitosan and basic amino acids. The method according to claim 1 or 2,
The composition is a composition for extending the lifespan of red blood cells, characterized in that compound K and decicinol are mixed at a weight ratio of 1 to 20: 1 to 20. delete A pharmaceutical composition for preventing or treating erythropoiesis, comprising the composition of claim 1 or 2 and a pharmaceutically acceptable excipient. The method of claim 5,
The erythropoietin is a erythropoiesis, pancony syndrome, granulocytopenia, myelodysplasia, aplastic anemia, hemolytic anemia, or iron deficiency anemia. A health functional food for improving red blood cell deficiency, comprising the composition of claim 1 or 2 and a food-acceptable food supplement additive. A composition for preserving blood comprising compound K and decursinol as active ingredients. The method of claim 8,
The blood preservation composition is a compound for preservation of blood, characterized in that it comprises compound K, decursinol, surfactant, heat-treated soluble chitosan and basic amino acids. In claim 8 or 9,
The blood preservation composition is a blood preservation composition characterized in that it is contained in the blood compound K 0.02 ~ 50μM and decursinol 10 ~ 50μM.

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