KR19990062375A - Compositions for Promoting Proliferation of Hematopoietic Stem Cells and Platelet Progenitor Cells Purified from Deer Antlers and Purification Methods Thereof - Google Patents

Abstract

It was confirmed that there is a strong active ingredient that promotes proliferation of hematopoietic stem cells in deer antler (plum), and the active ingredient having a hematopoietic stem cell growth promoting action was isolated and purified. In addition, the structure of the isolated and purified active ingredient was analyzed, and the active ingredient was synthesized. The synthesized active ingredient showed not only hematopoietic stem cell growth promoting effect but also platelet progenitor (megakaryocyte) proliferation effect.

As a method of detecting the effect of the hematopoietic stem cell growth promoting effect, a method of measuring the in vitro colony forming efficacy of hematopoietic stem cells is obtained. (The hematopoietic stem cells are collected and exposed to the separated and purified material. Observation), and silica gel column chromatography, TLC, and the like were used to separate the active ingredient from the antler.

Description

Compositions for Promoting Proliferation of Hematopoietic Stem Cells and Platelet Progenitor Cells Purified from Deer Antlers and Purification Methods Thereof

The present invention relates to a method for isolating and purifying an active ingredient having a hematopoietic stem cell growth promoting activity in antler (Plum), and a substance purified by the method. In particular, the present invention relates to a substance that promotes the growth of hematopoietic stem cells and platelet progenitor cells among the components of antler.

Originally, the deer antler is dried by collecting unhorned shells of deer belonging to the family Cornu Cervi, and is one of the most widely used herbal medicines with ginseng in Chinese medicine.

Deer antler has been widely used for thousands of years in Korea and other Northeast Asian regions. Among them, the deer antler origin animal is Cervus nippon Temminick var. Mantchuricus Swinhoe and Cervus elaphus. L. Cornu Ceriv Pavum).

Deer antler has various effects such as tonic action, growth and development promotion, hematopoietic action, neurodebilitating action, heart failure treatment action, and intestinal dysfunction of the five intestines. The effects of improving myocardial exercise, fatigue recovery, physical resistance enhancement, and pectoral angular effect have been recorded in ancient literature. Studies on the efficacy of antler include growth promoting action, hematopoietic action, serum cholesterol lowering action, protein synthesis promotion action, anti-ulcer effect, anti-aging effect, analgesic effect, anti-fatigue effect, immune activity enhancing effect and sedative effect. Reported. In addition, when lyophilized powdered antler was administered to animals in animal experiments, it reported strong analgesic effect, anti-fatigue effect against immobilization stress, weak immune activity enhancement effect and sedative effect.

Studies on the components of the deer antler revealed the composition of free amino acids and trace elements, and the composition of hexasaccharide, pentose, hexamine, uronic acid, sialic acid, acid mucopolysaccharides, hyaluronic acid, chondroitin sulfate A, and fatty acids. , Prostaglandins and the like, the composition of glycolipids and phospholipids, cholesterol, hypoxanthine, cholester-5-ene-3β, 7α-diol, cholesterol esters, polyamines, etc. have been reported separately. Estrone and estradiol receptors are known to contain.

As a medicinal herb medicinal roughly look as follows. Deer antler accelerates growth, stimulates hematopoietic function, increases blood cell production, and promotes proliferation of bone marrow cells. It also affects gonadotropin, increasing estrogen secretion and stimulating nucleic acid metabolism in the testes. In the heart and vascular system, a large amount can cause capillaries to expand, lower blood pressure, narrow the contraction amplitude of the heart, and slow the heart rate. However, despite the excellent efficacy of antler, the structure of the component showing the efficacy has not yet been found.

The present inventors have done a lot of research to find a component having an effect on hematopoiesis in antler known to have excellent medicinal effects as described above.

Currently, cancer disease, the number one cause of death in Korea, has been found to increase every year. Chemotherapy and radiation therapy used for chemotherapy damage not only cancer cells, but also normal normal bone marrow cells, especially hematopoietic cells that regulate immune and hematopoietic functions, thereby impairing hematopoietic organs and immune functions in the human body. Paralysis has been pointed out as a serious problem. This severe bone marrow cell damage leads to death of immune function paralysis resulting from reduced white blood cells or platelets. Therefore, malignant solid cancer, malignant blood disease, aplastic anemia, congenital intermittent disease, in which a condition requiring reduced immune function and blood cell function by promoting growth of hematopoietic stem cells, in particular, bone marrow function is reduced after anticancer drugs and irradiation There is an urgent need for the development of new substances that can be used in various clinical areas, including leukopenia, congenital immunity and hematopoietic cell deficiency, anemia caused by end stage renal failure, infection caused by leukocyte reduction, and myelosuppression due to aging population. It is requested.

As the research on hematopoietic stem cells has been actively conducted for the improvement of immune function and hematopoietic function, recently, 2-3 kinds of hematopoietic stem cell growth promoting factors (GM-CSF; granulocyte macrophage-colony) The stimulating factor, G-CSF (granulocyte-colony stimulating factor, Erythropoietin) was successfully commercialized. GM-CSF and G-CSF induce proliferation and differentiation of hematopoietic stem cells, as well as granulocytes and megakaryocytes, and promote mobilization of hematopoietic stem cells from bone marrow to peripheral blood.

In order to find such hematopoietic stem cell growth promoters, the present inventors have studied the antler, which has been used as a tonic tonic for thousands of years. As a result, the present inventors observed that a substance which promotes the growth of hematopoietic stem cells is present in the component of antler. Through continuous research, the most potent ingredient was found in antler, and the component was isolated and purified. And the structure of the component was revealed. After the structural analysis of the active ingredients from the inventors' study, it will be possible to develop new drugs by synthesizing synthetic materials. This new drug could be used to treat many incurable diseases, blood and immune diseases. In addition, the development of hematopoietic stem cell promoters may greatly contribute to the development of clinical fields such as hematology, oncology, immunology, infectious science and nephrology.

Until recently, the present inventors have systematically identified the immunobiological effects on the hematopoietic system among the various physiological activities of the plum extract and have not isolated the active ingredients.

Therefore, an object of the present invention is to find a hematopoietic stem cell promoter in antler and to reveal its structure. These hematopoietic stem cell growth promoting factors are diseases that require the growth of hematopoietic stem cell growth to promote immune function and the function of blood cells, in particular, malignant solid cancer, malignant blood disease, It can be used in various clinical fields such as aplastic anemia, congenital intermittent leukopenia, congenital immunity and hematopoietic cell deficiency, anemia caused by end stage renal failure, infection due to leukocyte reduction, and myelo function due to aging population. .

1 shows a chromatogram of high performance TLC of the purified component of CHCl ₃ extract of Deer Antler (Plum).

2 shows a positive FAB mass spectrum of CN-C-2-E-a-Mi.

Figure 3 shows a negative FAB mass spectrum of CN-C-2-E-a-Mi.

4 shows the tandem mass spectrum of the peak at m / z 577 of FIG.

5 shows the structure and molecular weight of C ₃₉ H ₇₂ O ₆ , the final separation material of the present invention. Where A represents the structure of 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol, and B represents the structure of 1-hexadecanoyl-2-9-octadecenoyl-3-acetylglycerol Indicates.

Figure 6 shows a spleen picture of the mouse administered the synthesized active material.

7 to 11 are microscopic findings showing the histological changes of the mouse spleens to which the synthesized active substance was administered.

Hematopoiesis is a series of developmental processes in which a large number of mature blood cells with very specific functions and a limited lifespan are produced and continuously supplied. The hematopoietic system consists of a very complex tissue system of hematopoietic precursor cells that can be aligned according to a system of developmental capacities. The source of mature blood cells of all lineages is the bone marrow, which contains cells at various stages of maturity, and the blood cells produced in the bone marrow are destroyed in peripheral tissues after some time, and this process is the life of the animal. It happens continuously through. Hematopoietic activity occurs in the embryonic yolk sac between 7 and 12 days and most active in the liver between 11 and 21 days, but also in the spleen and bone marrow. From the bone marrow. Hematopoietic action is maintained by hematopoietic stem cells (HSCs) that can proliferate and differentiate into cells of various lineages.

Initially, hematopoietic stem cells are called Pluripotent Hematopoietic Stem Cells (PHSCs), which go through a number of unknown processes to produce hematopoietic progenitor cells that can differentiate into a wide variety of mature blood cells. These hematopoietic progenitor cells produce the next, more differentiated cells.

Hematotopoietic Stem Cells (HSCs) have two main characteristics. It is the ability to produce self-renewal to produce the same cells as itself and to differentiate into all lines of mature lymphocytes. The study of hematopoietic stem cells began when the animal first discovered that the deficiency could be recovered by irradiating a sublethal dose of radiation and then injecting normal bone marrow cells into animals suffering from bone marrow deficiency. From the first quantitative experiments on bone marrow recovery of mice subjected to whole body radiation, hematopoietic progenitor cells (multipotent cells) with the ability to form bone marrow cells and erythroid cell colonies in the spleen and bone marrow of irradiated animals The concept of hematopoietic stem cell was introduced. In order to understand the reconstitution of hematopoietic cells by self-replicating hematopoietic stem cells and the developmental biological aspects of the hematopoietic system, isolation of pure hematopoietic stem cells is required. However, these cells were difficult to isolate because they exist at very low rates.However, the introduction of flow cytometry and the development of monoclonal antibodies against hematopoietic stem cell markers and differentiation antigens resulted in HSCs ( Hematopoietic Stem Cells) can be screened and their properties and characteristics gradually understood.

Representative methods for searching for efficacy on hematopoietic action include a method of measuring colony forming efficacy of collecting hematopoietic stem cells, exposing them to a search material, culturing them in vitro, and monitoring the formation of hematopoietic stem cell clones, and in vivo searching using animals. As a method, there is a method (colony forming unit in the spleen) for measuring the degree of recovery of the reduced bone marrow function after a subdivision dose or by administering a large amount of anticancer agents. Solvent extraction, silica gel chromatography, and the like are used to separate and purify the antler component, and MASS and NMR are used as the structural analysis methods.

Hereinafter, the present invention will be described by way of examples. The examples are illustrative only and do not limit the scope of the invention.

Example 1

1.75 kg of deer antler (plum) was added to a crushed beaker, hexane (approximately 6 liters) was added to the antler, and the mixture was heated at 80 ° C. for 2 hours, extracted twice, and filtered (this filtrate was called extract 1). . The extract 1 was distilled off under reduced pressure to obtain 24.6 g of extract (extract CN-H).

Chloroform was added to the residue to the degree of immersion (approximately 6 liters), heated at 70 ° C. for 2 hours, extracted twice, and filtered (this filtrate was called Extract 2). Extract 2 was distilled off under reduced pressure to give 14 g of extract (extract CN-C). 70% ethanol (about 6 liters) was added to the residue, and the mixture was heated at 90 ° C for 2 hours, extracted three times, and filtered (this filtrate was called extract 3). The extract 3 was distilled off under reduced pressure to obtain 91 g of an extract (extract CN-E).

Example 2

A chloroform / methanol (500: 1) mixed solution was added to 350 g of powdered silica gel, inflated, and filled into an open column (bed volume = 600 to 650 ml]. 12 g of extract CN-C was dissolved in a minimum amount (the minimum amount that could dissolve the extract) in chloroform / methanol (500: 1) and applied to a column filled with silica gel. Eluent chloroform / methanol (500: 1) was added 50 ml each while not drying (the volume of the void was 200 ml), and this was developed by TLC (developing solvent: chloroform / methanol = 300: 1). It was. It was developed with iodine and then separated according to Rf. At this time, there were seven main parts separated. The separated portions were each distilled under reduced pressure and dried. Each is represented by CN-C-1 to CN-C-7. Purification was continued as an indicator of hematopoietic stem cell proliferation activity.

Example 3

Screening of Hematopoietic Effect of Deer Antler

The present inventors performed the following experiment to search for the hematopoietic effect of the antler extract.

1) Isolation of Mouse Bone Marrow Cells

Bone marrow was isolated by washing with RPMI 1640 nutrient medium (Gibco # 430-1800) from the tibia and femur of the mouse leg. The separated bone marrow was filtered to make a single cell suspension, and again washed three times with RPMI 1640 nutrient medium, and the number of cells was measured. A stem cell rich fraction having a specific gravity between 1.063 and 1.075 was obtained and washed twice with RPMI.

2) Preparation of Sample

All antler samples were thawed to a concentration of 0.8 mg / ml DMSO, which was used as a stock solution and used to search for hematopoietic efficacy.

3) Conduct CFU-C (colony forming unit in culture) analysis

The cells isolated in the same manner as in 1) were planted in 1.1% methylcellulose plate at 1.2 × 10 ⁴ cells / well, then cultured in a 37 ° C. CO ₂ incubator, and after one week the number of colonies Measured.

Colony Forming Efficiency (CFE) = (total number of colonies in sample) / (total number of colonies in control)

The hematopoietic efficacy search experiments were performed on the antler extract samples from each example.

실험) Experimental animal

Male mice of 6 to 9 weeks old C57B1 / 6 breeding under SPF (Specific Pathogen free) conditions are used.

Ii) efficacy search

Bone marrow cells are isolated from the tibia and femur of the mouse hind limb by flushing with RPMI 1640 medium (Giboco # 430-1800). The isolated bone marrow is passed through a cotton filter into a single cell suspension. This was again washed with RPMI 1640 medium and the cell number was measured using a hemocytometer. The whole BM cells were subjected to discontinuous gradient centrifugation using Percoll solutions having densities of 1.090, 1.075, and 1.063 to obtain a stem cell-rich fraction and then washed. The parental-rich fractions obtained above measure the viability and number of cells using trypan blue dye exclusion method. Each antler sample is dissolved in 100% DMSO to a concentration of 0.8 mg / ml. Prepare a tube for each sample and concentration, add a certain amount of FBS, medium, methyl cellulose, cells, etc., mix two aliquots in a well plate and incubate for one week in a 5% CO ₂ incubator. After 1 week, stain and count colonies under an inverted microscope. Compared to the control, the activity (colony forming efficacy) is confirmed. The control here does not contain only the antler sample.

When the number of colonies of the control was 154, the extract CN-C was dissolved in the culture solution to a concentration of 0.001 mg / ml, and the number of colonies after incubation was 171. The colony forming efficacy here is 171/154 with 1.11 (control CFE = 1).

The above hematopoietic effect data are shown in the table below.

Sample Colony Formation Efficiency (0.001mg / ml Concentration) Number of colonies in sample / total colonies Extract CN-C 1.11 171/154 Extract CN-C-2 1.22 188/154 Extract CN-C-2-E 1.23 189/154 Extract CN-C-2-E-a 1.29 199/154 Extract CN-C-2-E-a-Mi 1.30 200/154

Example 4

In Example 2, CN-C-2 (380 mg), a fraction of high hematopoietic efficacy, was taken from the purified fraction. A mixed solution of hexane / ethyl acetate (50: 1) was added to 20 g of powdered silica gel, and it was inflated and filled in a column (bed volume = 100 ml). Extract CN-C-2 was dissolved in a minimum amount of eluent hexane / ethyl acetate (50: 1) and applied to a silica gel column. As in Example 2, while taking the eluent was added 15ml each was developed by TLC (developing solvent: hexane / ethyl acetate = 50: 1), after iodine coloration was separated into six parts according to Rf. The separated portions were each distilled under reduced pressure and dried. Each is represented by CN-C-2-A to -F.

Example 5

In the fraction purified in Example 4, CN-C-2-E (70 mg), which is a fraction with high hematopoietic effect, was taken. A mixed solution of hexane / ethyl acetate / acetic acid (20: 1: 0.5) was added to 3.5 g of powdered silica gel, and it was inflated and filled in a column (volume = 24 ml). Extract CN-C-2-E was dissolved in a minimum amount of hexane / ethyl acetate / acetic acid (20: 1: 0.5) as an eluent and applied to a silica gel column. 10 ml each was added while the eluent was added as in Example 3, and developed by TLC (developing solvent: hexane / ethyl acetate / acetic acid = 20: 1: 0.5), and was divided into two parts according to Rf after iodine coloration. The divided portions were dried by distillation under reduced pressure, respectively. Two parts are marked a and b.

Example 6

The first (extract CN-C-2-E-a) part of the part divided in Example 5 was taken. After mixing 5 ml of methanol in extract CN-C-2-Ea, mix and dissolve in methanol (extract CN-C-2-Ea-Ms) and insoluble in methanol (extract CN-C-2-Ea-Mi). Separated. The extract CN-C-2-E-a-Ms obtained 20 mg, and the extract CN-C-2-E-a-Mi obtained 40 mg.

The TLC results of Examples 2, 4, and 5 are shown in FIG. As shown in Figure 1, it can be seen that the band in the extract CN-C appears the same in the process of purification. Therefore, it can be seen that the part showing activity is not denatured or disappeared during purification. At first, the band, which was covered by other parts, developed deeper as it was refined.

Example 7

Structural analysis of hematopoietic active substances

Extract CN-C-2-E-a-Mi was analyzed in a VG70-VSEQ mass spectrometer among the two substances finally purified from Example 6, and is shown in FIG. 2. The analysis method was low resolution and positive fast atom bombardment (FAB) was used as the ionization method, and 3-nitro benzyl alcohol was used as a matrix.

The molecular ion M ⁺ is 637.5 and m / z 577.5 is 3-deoxy-1-oleoyl-2-palmitoyl glyceride-3-carbo cation without acetic acid (CHCOOH). 2-palmitoyl glyceride-3-carbocation), m / z 383.3 is the component from which the palmitate group has been removed, and m / z 239.2 represents the palmitoyl group.

3 shows the results of negative fast atom bombardment (FAB) of extract CN-C-2-E-a-Mi. m / z 255.2 is a palmitate anion and m / z 281.3 is an oleate anion.

4 is a tandem mass spectrum for m / z 577 of FIG. m / z 393.3 is a structure in which -C ₁₃ H ₂₇ group and proton are removed from 3-deoxy-1-oleyl-2-palmitoyl glyceride-3-carbo cation, and m / z 367 is- C ₁₅ H ₂₉ and protons removed.

As a result of analyzing the mass spectrum of FIGS. 2 to 4, the structure of the purified two components was 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol (1-9-octadecenoyl-2-hexadecanoyl- 3-acetylglycerol) and 1-hexadecanoyl-2-9-octadecenoyl-3-acetylglycerol (1-hexadecanoyl-2-9-octadecenoyl-3-acetylglycerol), the structure of which is shown in FIG.

Example 8

Synthesis of Hematopoietic Stem and Platelet Progenitor Proliferation Factors

A. Synthesis of 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol: acetolysis of phosphatidylcholine

2-5 mg of phosphatidylcholine (1-9-octadecenoyl-2-hexadecanoyl-3-phosphocholine) was dissolved in 1 ml of acetic acid: acetic anhydride (3: 2) and placed in a glass vial. The vial was tightly tied and deeply reacted in an oil bath at 150 ° C. for 8 hours. After the reaction, the mixture was extracted three times with chloroform: methanol: water (8: 4: 3), washed with water, and dried over anhydrous sodium sulfate for 1 hour. The structure of the composite was confirmed by TLC, GC-MS and NMR.

B. Synthesis of 1-hexadecanoyl-2-9-octadecenoyl-3-acetylglycerol: isolation of phosphatidylcholine from egg yolk

40 ml of ether was added to one egg yolk and stirred for about 1 hour. After evaporating the solvent in the water bath with only the upper part of the melted water, 30 ml of acetone is added and stirred, and the pure phosphatidylcholine sinks into the precipitate. This precipitate was dissolved in chloroform: methanol: water (65: 25: 4) as eluent. Powder silica gel was poured into chloroform: methanol: water (65: 25: 4), expanded, and filled into an open column. A non-pure phosphatidylcholine precipitate was applied to the packed column and fractions were collected by 20 ml. Only pure phosphatidylcholine (1-hexadecanoyl-2-9-octadecenoyl-3-phosphocholine) portion was collected by TLC compared to the standard phosphatidylcholine. Acetolysis of the collected material was carried out in the same manner as in A to synthesize the desired material.

Example 9

In vivo efficacy analysis using synthetic materials (in vivo CFU-S analysis)

C57B1 / 6 mice aged 6 to 10 weeks were irradiated with 8.0Gy at a 137-Cs irradiator (CIS #IBL 437) and irradiated with 1.5G10 ⁵ allogeneic bone marrow cells according to Till McCulloch's method. Bone marrow transplantation was performed through the tail vein. Mice were divided into control group and synthetic group. Each factor was injected on day 1, day 2, and day 3 after bone marrow transplantation, and spleens were extracted on day 14, and colony formation was observed and fixed with formalin. At this time, nodules (nodules) formed in the spleen were compared and observed.

The results are shown in FIG.

In Figure 6, A is a control, B is a antler water-soluble extract, C is a synthetic 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol (1 mg, 3 Daily administration), D is a synthetic 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol (0.1 mg, 3 days), and E is a synthetic 1-9-octadeceno It was administered 2--2-hexadecanoyl-3-acetylglycerol (0.01 mg, 3 days administration).

6 shows that the spleen of the mouse administered with synthetic 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol than the spleen of the mouse administered the control group (A) and the antler water-soluble extract (B). It can be seen that this size is larger and more nodules are produced, and as the dosage of the synthetic material increases, the spleen increases in size and more nodules occur (E <D <C).

From the results of FIG. 6, the hematopoietic stem cell proliferation promoting activity of the synthetic material can be confirmed.

Example 10

Microscopic Findings of the Mouse Spleen

The 6-10 weeks of age by the C3H / He mice using a 137 Cs-radiation emitter (irradiator) (CIS #IBL 437) after whole body irradiated with 8.0Gy radiation amount according to the method of Till McCulloch isolated from allogeneic mice, 1.5 x 10 ⁵ Bone marrow cells were suspended in RPMI medium and injected into the tail vein. At 1, 2 and 3 days after allogeneic bone marrow transplantation, the control group was intraperitoneally injected with 100 μl of sterile PBS (1% BSA). 100 μl each was injected intraperitoneally. At 14 days after allogeneic bone marrow transplantation, the spleen was extracted, fixed in 10% PBS buffered formalin solution, and cut and subjected to Hematoxylin / Eosin staining.

The results are shown in Figures 7-11.

7 is a spleen photograph at low magnification (40 ×) and high magnification (400 ×) of the control group administered with phosphate buffer only, and the spleen is atrophyed as a whole. Figure 8 is a micrograph of the group to which the antler soluble extract was administered. The proliferation of hematopoietic stem and progenitor cells can be observed in the white pulp of the spleen.

Figure 9 is a micrograph of a group administered with 1 mg of the synthetic substance 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol. At low magnification (40X), the white water quality of the spleen is increased, and at high magnification (100X and 400X), proliferation of hematopoietic stem cells and hematopoietic stem cell progenitor cells and platelet progenitor cells (megakaryocytes) are interspersed.

10 is a micrograph of a group administered with 0.1 mg of the synthetic substance 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol. In the white medulla of the spleen, hematopoietic stem cell proliferation and platelet progenitor cells can be observed.

Figure 11 is a micrograph of the group administered with 0.01 mg of the synthetic material 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol. The expansion of the white medulla of the spleen and the clusters of hematopoietic stem, progenitor and platelet progenitor cells can be observed.

7 to 11, the proliferation promoting activity of hematopoietic stem cells and platelets of the synthetic material can be confirmed.

The present inventors isolated and purified hematopoietic stem cells and platelet proliferation promoters from antler, and discovered the structure of the substance. The novel substance according to the present invention has a role in promoting hematopoietic stem cell and platelet proliferation, and in various clinical fields, such as a disease in which immune function and blood cell function are required to be enhanced, in particular, a condition in which bone marrow function is degraded after anticancer drugs and irradiation. Can be used as a therapeutic agent throughout.

Claims (6)

A composition for promoting hematopoietic cell growth or platelet growth, comprising an extract of antler extracted with a solvent selected from the group consisting of hexane, chloroform and ethanol as an active ingredient. The composition of claim 1, wherein the extract is further purified by silica gel column chromatography and TLC. The composition according to claim 1 or 2, wherein the extract comprises 1-9-octadecenoyl-2-hexadecanoyl-3-acetylglycerol. The composition according to claim 1 or 2, wherein the extract comprises 1-hexadecanoyl-2-9-octadecenoyl-3-acetylglycerol. Extract filtration of the antler with a solvent selected from the group consisting of hexane, chloroform and ethanol; Distilling the extract filtrate under reduced pressure to obtain an extract; Then applying the extract to a silica gel column; Eluting the silica gel column and then developing the fractions obtained by TLC; And separating it according to Rf after color development of iodine. The method of extracting and purifying hematopoietic growth factor or platelet growth promoting factor in antler. 6. The method of claim 5, wherein the silica gel column is eluted with an eluent selected from the group consisting of chloroform / methanol, hexane / ethyl acetate and hexane / ethyl acetate / acetic acid.